Correlative imaging using super-resolution fluorescence microscopy and soft X-ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development.

Active virosomes (AVs) are derivatives of viruses, broadly similar to 'parent' pathogens, with an outer envelope that contains a bespoke genome coding for four to five viral proteins capable of eliciting an antigenic response. AVs are essentially novel vaccine formulations that present on their surface selected viral proteins as antigens. Once administered, they elicit an initial 'anti-viral' immune response. AVs are also internalised by host cells where their cargo viral genes are used to express viral antigen(s) intracellularly. These can then be transported to the host cell surface resulting in a second wave of antigen exposure and a more potent immuno-stimulation. A new 3D correlative microscopy approach is used here to provide a robust analytical method for characterisation of Zika- and Chikungunya-derivatised AV populations including vesicle size distribution and variations in antigen loading. Manufactured batches were compared to assess the extent and nature of batch-to-batch variations. We also show preliminary results that verify antigen expression on the surface of host cells. We present here a reliable and efficient high-resolution 3D imaging regime that allows the evaluation of the microstructure and biochemistry of novel vaccine formulations such as AVs.

A novel combination of microscopies involving X-ray and laser light has been developed at the correlative cryo-imaging beamline B24 of the UK synchrotron which can be used to analyse across- and within-batch variability of active virosome vaccine formulations. We use 3D fluorescence imaging to localise viral components within vaccine vesicles and soft X-ray tomography to characterise sample variability and impact upon delivery to cells. Moreover, we offer the next step in automation of data processing and evaluation to further enable rapid assessment of exosome-based vaccines. Active virosome vaccines are suspensions of membrane-bounded vesicles that carry antigens and genetic material from select viral pathogens. These elicit both an initial immune response through their introduction and a subsequent sustained antigenic potential via gene expression in host cells. In this case, as in all novel vaccine formulations, rapid assessment and batch standardisation are of paramount importance for the medical community and the methods described here provide a robust way of quick and efficient assessment and validation of formulations during research and development and at the production stages.

Tunable Chiral Second-Order Nonlinear Optical Chromophores Based on Helquat Dications.

Fourteen new dipolar cations have been synthesized, containing methoxy or tertiary amino electron donor groups attached to helquat (Hq) acceptors. These Hq derivatives have been characterized as their TfO- salts by using various techniques including NMR and electronic absorption spectroscopies. UV-vis spectra show intense, relatively low energy absorptions with λmax ≈ 400-600 nm, attributable to intramolecular charge-transfer (ICT) excitations. Single-crystal X-ray structures have been solved for two of the chromophores, one as its PF6- salt, revealing centrosymmetric packing arrangements (space groups Pbca and P1̅). Molecular quadratic nonlinear optical (NLO) responses have been determined directly by using hyper-Rayleigh scattering (HRS) with a 800 nm laser, and indirectly via Stark (electroabsorption) spectroscopy for the low energy absorption bands. The obtained static first hyperpolarizabilities ß0 range from moderate to large: (9-140) × 10-30 esu from HRS in MeCN and (44-580) × 10-30 esu from the Stark data in PrCN. The magnitude of ß0 increases upon either extending the π-conjugation length or replacing a methoxy with a tertiary amino electron donor substituent. Density functional theory (DFT) and time-dependent DFT calculations on selected tertiary amino chromophores confirm that the low energy absorptions have ICT character. Relatively good agreement between the simulated and experimental UV-vis absorption spectra is achieved by using the CAM-B3LYP functional with the 6-311G(d) basis set. The ßtot values predicted by using DFT at the same level of theory are large ((472-1443) × 10-30 esu in MeCN). Both the theoretical and experimental results show that para-conjugation between Hq and electron donor fragments is optimal, and enlarging the Hq unit is inconsequential with respect to the molecular quadratic NLO response.

Helquat Dyes: Helicene-like Push-Pull Systems with Large Second-Order Nonlinear Optical Responses.

Helquat dyes combine a cationic hemicyanine with a helicene-like motif to form a new blueprint for chiral systems with large and tunable nonlinear optical (NLO) properties. We report a series of such species with characterization, including determination of static first hyperpolarizabilities ß0 via hyper-Rayleigh scattering and Stark spectroscopy. The measured ß0 values are similar to or substantially larger than that of the commercial chromophore E-4'-(dimethylamino)-N-methyl-4-stilbazolium. Density functional theory (DFT) and time-dependent DFT calculations on two of the new cations are used to probe their molecular electronic structures and optical properties. Related molecules are expected to show bulk second-order NLO effects in even nonpolar media, overcoming a key challenge in developing useful materials.

Ferrocenyl helquats: unusual chiral organometallic nonlinear optical chromophores.

Three new dipolar cations have been synthesised, containing ferrocenyl (Fc) electron donor groups attached to helquat (Hq) acceptors. These organometallic Hq derivatives have been characterised as their TfO- salts by using various techniques including NMR and electronic absorption spectroscopies and electrochemical measurements. UV-vis spectra show multiple intense low energy absorptions attributable to intramolecular charge-transfer (ICT) excitations. Each compound displays a reversible Fc+/0 redox process, together with two reversible one-electron reductions of the Hq fragment. Molecular quadratic nonlinear optical (NLO) responses have been determined by using hyper-Rayleigh scattering at 1064 nm, and Stark (electroabsorption) spectroscopic studies on the visible absorption bands. The obtained first hyperpolarizabilities ß are moderate, consistent with the relatively short π-conjugation lengths between the Fc and attached pyridinium group. A single-crystal X-ray structure has been solved for one of the complexes as its PF6- salt, revealing a centrosymmetric packing in the triclinic space group P1[combining macron]. Density functional theory (DFT) and time-dependent DFT calculations indicate that the lowest energy absorption bands have mainly metal-to-ligand charge-transfer character. The donor orbitals involved in the electronic transitions forming the next lowest energy ICT band also have substantial contributions from the Fe atom. Good agreement between the simulated and experimental UV-vis absorption spectra is achieved by using the PBE0 functional with the 6-311++G(d)/LANL2DZ mixed basis set, and the theoretical ß values are reasonably large. Oxidation of the Fc unit is predicted to cause the ßtot value to decrease by more than 80% in one of the complexes.

A role for Stat1 in the regulation of lipopolysaccharide-induced interleukin-1beta expression.

Because the induction of interleukin-1beta (IL-1beta) is critical to antibacterial host defenses and its excessive generation is a prominent component of sepsis, regulation of this proinflammatory cytokine is a critical factor in the immune response to lipopolysaccharide (LPS). We previously showed that LPS-induced IL-1beta expression was regulated by a Stat1-dependent, nitric oxide (NO)-mediated mechanism. Subsequent in vivo studies showed that whereas Stat1 had a role in the downregulation of IL-1beta expression, it had a more significant effect on its initial induction. Although both interferon-beta (IFN-beta) and IFN-gamma activate Stat1, the early appearance of IFN-beta in the circulation after LPS administration suggested its pivotal role in Stat1-mediated IL-1beta expression in vivo. Further in vitro analysis of peritoneal macrophages from IFN-beta (/), Stat1(/), and caspase-1(/) mice and their wild-type controls following LPS stimulation demonstrated that IL-1beta mRNA was expressed in these mice but not in macrophages from MyD88(/) mice. Despite the presence of IL-1beta mRNA, IL-1beta protein was markedly reduced in the absence of Stat1 activation in macrophages derived from IFN-beta (/) and Stat1(/) mice or in the absence of caspase-1 activity, which itself was dependent on Stat1 activation. These studies support the hypothesis that the expression of IL-1beta requires both the MyD88-dependent induction of IL-1beta mRNA and pro-IL-1beta as well as the MyD88-independent, Stat1-mediated processing of that gene product into active cytokine.

Simultaneous activation of apoptosis and inflammation in pathogenesis of septic shock: a hypothesis.

Sepsis, a widely prevalent disease with increasing morbidity and mortality, is thought to result from uncontrolled inflammatory responses to microbial infection and/or components. However, failure of several experimental anti-inflammatory therapies has necessitated re-evaluation of the paradigm underlying the pathogenesis of this complex disorder. Apoptotic cell death forms a second dominant feature of septic shock in patients and animal models. Anti-apoptotic strategies may protect animals from septic death. However, simultaneous occurrence of apoptosis and inflammation is necessary for septic death. At the cellular level, apoptosis plays a central role in the development of the lymphoid system and regulation of immune responses. Immune activation renders cells refractory to apoptosis while apoptosis of activated lymphocytes is an important immunoregulatory mechanism. Factors such as complement factor 5a, caspase-1 and mitogen-activated protein kinase, which participate in apoptosis as well as pro-inflammatory pathways, may be responsible for simultaneous activation of apoptosis and inflammation in sepsis. Further identification of other similar biochemical events capable of co-activating inflammation and apoptosis may provide new targets for therapy of this hitherto untreatable disease.

Lipopolysaccharide-induced paw edema model for detection of cytokine modulating anti-inflammatory agents.

Cytokines are critical to pathogenesis of inflammatory disorders. So inhibition of their action provides therapeutic benefits in various diseases. Although inhibition of inflammation caused by intraperitoneally administered LPS can identify cytokine modulators, this inflammatory test-agent does not allow one to determine overall anti-inflammatory potential. Functional characteristics of Carrageenan (Cara)-induced edema were valuable for identification of nonsteroidal anti-inflammatory drugs (NSAIDS). Hence, the potential of LPS-induced paw inflammation was investigated and compared to that by Cara. Stimulation of isolated rat peritoneal exudates cells (PEC) with 10 ng/ml LPS, but not Cara, induced IL-6 (3.04+/-0.2 ng/ml) and TNFalpha (1.030.09 ng/ml). At least 100 mg/ml Cara was necessary for detection of IL-6 (2.03+/-0.1 ng/ml) and TNFalpha (0.6+0.09 ng/ml) in PEC. Similar to Cara, subplantar administration of LPS-induced inflammatory paw edema in rats. LPS, but not Cara, induced TNFalpha (2.14+/-0.6 ng/ml) and IL-6 (2.9+/-0.5 ng/ml) in serum at 1 and 3 h, respectively, which returned to basal levels by 5 h. LPS-induced serum TNFalpha (sTNFalpha) levels closely paralleled paw swelling and its neutralization by anti-TNFalpha antibody or inhibition by pentoxifylline and nimesulide correlated with inhibition of inflammation. Similar to earlier reports, rofecoxib induced sTNFalpha at 30 mg/kg and exhibited pro-inflammatory effect by enhancing paw swelling. LPS-induced edema provides a useful functional model for identification of cytokine modulating anti-inflammatory agents.

Role of caspase 1 in murine antibacterial host defenses and lethal endotoxemia.

Sepsis is thought to result from an exaggerated innate immune response to microbial components such as lipopolysaccharide (LPS), but the involvement of a specific mechanism(s) has not been identified. We studied the role of caspase 1 (Cas-1) in the murine innate immune response to infection with gram-negative bacteria and to nonlethal and lethal doses of LPS. cas-1(-/-) and Cas-1 inhibitor (Ac-YVAD-CHO)-treated cas-1(+/+) mice were two- to threefold more susceptible to lethal Escherichia coli infection than cas-1(+/+) mice. Administration of Cas-1 products, interleukin-18 (IL-18) or IL-1beta, protected three of three and six of seven mice, respectively, from lethal infection with E. coli compared to none of six of untreated mice (P = 0.0082). Therefore, cas-1 is essential for antibacterial host defense. Nonlethal (75 micro g) and lethal (500 micro g) doses of LPS induce different patterns of gamma interferon, IL-1beta, and IL-18 expression. Consequently, the role of Cas-1, which cleaves pro-IL-18 and pro-IL-1beta to their active forms, was investigated in these disparate conditions by using enzymatic assay and reverse transcription-PCR. At 75 micro g, LPS induced a transient increase in IL-1beta and IL-18 levels in serum, whereas at 500 micro g it induced a 1.5-fold-higher IL-18 level in serum, which increased till death. At 75 micro g of LPS, splenic cas-1 mRNA expression remained unchanged at all time points, but activity increased transiently at 3 h. In lethally treated mice, Cas-1 activity remained elevated until death; however, cas-1 mRNA levels increased at 3 h and decreased to basal levels by 8 h. Treatment with Cas-1 inhibitor protected mice from lethal endotoxemia. Thus, Cas-1 is essential for innate antibacterial host defenses and may represent a mechanism of innate immunity that upon excessive stimulation by microbial components may lead to endotoxic shock.

IL-18 levels and the outcome of innate immune response to lipopolysaccharide: importance of a positive feedback loop with caspase-1 in IL-18 expression.

LPS enhanced antibacterial host defenses (ABHD) when given at low (75 micro g) doses (16 of 19 mice survived 3x LD(50) Escherichia coli vs 3 of 19 LPS-naive mice; p = 0.0001), but induced lethal inflammation at high (500 micro g) doses (5 of 5 died). Differences in the cytokine profiles induced by these LPS doses may provide insight into the mechanism(s) of transition from beneficial to lethal LPS responses. The 75 micro g LPS induced 5.9 +/- 0.9 ng/ml serum IL-18 at 8 h, which decreased to 2.3 +/- 0.4 ng/ml by 24 h, whereas 500 micro g LPS induced 11.1 +/- 1.6 ng/ml serum IL-18 levels at 8 h, which increased until death. Compared with 75 micro g, higher but sublethal (150 micro g) doses of LPS induced greater serum IL-18 levels and less effectively induced ABHD (3 of 8 survived). Reduction of serum IL-18 with neutralizing Ab improved the ABHD induced by 150 micro g, but reduced that produced by 75 micro g LPS, suggesting an optimal range of serum IL-18 level was essential for efficient ABHD. Increased expression of caspase-1 mRNA in response to the higher IL-18 levels induced at the 150 and 500 micro g, but not at the 75 micro g doses of LPS may represent a positive feedback regulatory loop leading to sustained serum IL-18 levels. We conclude that the regulation of serum IL-18 expression is critical to the outcome of innate immune responses to LPS.