Lysosome directed red light photodynamic therapy using glycosylated iron-(III) conjugates of boron-dipyrromethene.

To overcome the drawbacks associated with chemotherapeutic and porphyrin-based photodynamic therapy (PDT) agents, the use of BODIPY (boron-dipyrromethene) scaffold has gained prominence in designing a new generation of photosensitizers-cum-cellular imaging agents. However, their poor cell permeability and limited solubility in aqueous medium inhibits the in-vitro application of their organic form. This necessitates the development of metal-BODIPY conjugates with improved physiological stability and enhanced therapeutic efficacy. We have designed two iron(III)-BODIPY conjugates, [Fe(L1/2)(L3)Cl] derived from benzyl-dipicolylamine and its glycosylated analogue along with a BODIPY-tagged catecholate. The complexes showed intense absorption bands (ε âˆ¼ 55,000 M-1 cm-1) and demonstrated apoptotic PDT activity upon red-light irradiation (30 J/cm2, 600-720 nm). The complex with singlet oxygen quantum yield value of ∼0.34 gave sub-micromolar IC50 (half-maximal inhibitory concentration) value (∼0.08 µM) in both HeLa and H1299 cancer cells with a photocytotoxicity index value of >1200. Both the complexes were found to have significantly lower cytotoxic effects in non-cancerous HPL1D (human peripheral lung epithelial) cells. Singlet oxygen was determined to be the prime reactive oxygen species (ROS) responsible for cell damage from pUC19 DNA photo-cleavage studies, 1,3-diphenylisobenzofuran and SOSG (Singlet Oxygen Sensor Green) assays. Cellular imaging studies showed excellent fluorescence from complex 2 within 4 h, with localization in lysosomes. Significant drug accumulation into the core of 3D multicellular tumor spheroids was observed within 8 h from intense in-vitro emission. The complexes exemplify iron-based targeted PDT agents and show promising results as potential transition metal-based drugs for ROS mediated red light photocytotoxicity with low dosage requirement.

Dynamic changes in thymic sub-populations during acute and long-term infections with virulent and virulence-attenuated Salmonella Typhimurium strains in C57BL/6 and autoimmune-prone lpr mice.

SALMONELLA: Typhimurium infection in mice results in drastic loss of immature CD4- CD8- double negative (DN) and CD4+ CD8+ double positive (DP) thymic subsets compared to mature single positive (SP) subsets. We investigated changes in thymocyte sub-populations post infection with a wild type (WT) virulent strain and ΔrpoS, a virulence-attenuated strain, of Salmonella Typhimurium in C57BL/6 (B6) and Fas-deficient autoimmune-prone lpr mice. The WT strain caused acute thymic atrophy with greater loss of thymocytes in lpr mice compared to B6 mice. Infection with ΔrpoS caused progressive thymic atrophy in B6 and lpr mice. Analysis of thymocyte subsets revealed that immature thymocytes including the DN, immature single positive (ISP), and DP thymocytes underwent extensive loss. SP thymocytes were more resistant to loss in WT-infected B6 mice, whereas WT-infected lpr and ΔrpoS-infected mice exhibited depletion of SP thymocytes. Overall, thymocyte sub-populations exhibited differential susceptibilities depending on bacterial virulence and the host background.

Bichromophoric ruthenium(II) bis-terpyridine-BODIPY based photosensitizers for cellular imaging and photodynamic therapy.

Two multichromophoric homoleptic ruthenium(II) complexes [Ru(tpy-BODIPY)2]Cl2 (complexes 1 and 2, tpy = 4-phenyl-2,2:6,2-terpyridine, BODIPY = boron-dipyrromethene) were prepared, characterized and their phototherapeutic activity and bioimaging properties were studied. The complexes having structural similarity differ only by a phenylethynyl linker, and its overall influence on their physicochemical and photobiological behavior was evaluated. The terpyridine-BODIPY ligand L1 was structurally characterized by X-ray crystallography. The complexes showed intense absorption near 500 nm (ε: ∼1.5 × 105 M-1 cm-1 in DMSO), have a high singlet oxygen quantum yield (ΦΔ: ∼0.6 in DMSO), and displayed low photobleaching thus making them suitable for PDT applications. The complexes showed high DNA binding affinity and induced DNA damage on light activation via multiple types of ROS production. Confocal laser scanning microscopy experiments revealed their incorporation in the cancer cells and complex 1 predominantly accumulated in lysosomes. The complexes displayed a significant PDT effect in cancerous cells with visible light activation with a high photocytotoxicity index (PI) value in HeLa cells. Both type-I and type-II photosensitization processes were involved in the PDT effect. The photodynamic action of complex 2 initiated cellular apoptosis. Finally, their diagnostic potential was evaluated against clinically relevant 3D multicellular tumor spheroids (MCTs).

Countries with high deaths due to flu and tuberculosis demonstrate lower COVID-19 mortality: roles of vaccinations.

Deaths due to the ongoing COVID-19 pandemic vary (3-1681 deaths/million and mortality rates 0.71-14.54%) and are far greater in some countries compared to others. This observation led us to perform epidemiological analysis, using data in the public domain, to study the correlation of COVID-19 with the prevalence and vaccination strategies for two respiratory pathogens: flu and tuberculosis (TB). Countries showing more than 1000 COVID-19 deaths were selected at three time points during the ongoing pandemic: 17 May, 1 October and 31 December 2020. The major findings of this study that are broadly consistent at all three time points are: First, countries with high flu deaths negatively correlate with COVID-19 deaths/million. Second, TB incidences and deaths negatively correlate with COVID-19 deaths/million. Countries displaying high TB and flu deaths (Nigeria, Ethiopia, Myanmar, Indonesia, India) display lower COVID-19 deaths/million compared to countries with low TB and flu deaths (Italy, Spain, USA, France). Third, countries with greater flu vaccination display lower flu incidences but higher COVID-19 deaths/million and mortality rates. On the other hand, Bacillus Calmette Guerin (BCG) vaccination negatively correlates with Covid-19 deaths/million. Fourth, countries with only BCG, but no flu, vaccination show delayed and lower number of COVID-19 deaths/million compared to countries with flu, but no BCG, vaccination. Fifth, countries with high BCG vaccination coverage as well as high TB deaths display the lowest COVID-19 deaths/million. The implications of this global study are discussed with respect to the roles of respiratory infections and vaccinations in lowering COVID-19 deaths.

7-Hydroxy Frullanolide, a sesquiterpene lactone, increases intracellular calcium amounts, lowers CD4+ T cell and macrophage responses, and ameliorates DSS-induced colitis.

Sesquiterpene lactones are a class of anti-inflammatory molecules obtained from plants belonging to the Asteraceae family. In this study, the effects of 7-hydroxy frullanolide (7HF), a sesquiterpene lactone, in inhibiting CD4+ T cell and peritoneal macrophage responses were investigated. 7HF, in a dose dependent manner, lowers CD69 upregulation, IL2 production and CD4+ T cell cycling upon activation with the combination of anti-CD3 and anti-CD28. Further mechanistic studies demonstrated that 7HF, at early time points, increases intracellular Ca2+ amounts, over and above the levels induced upon activation. The functional relevance of 7HF-induced Ca2+ increase was confirmed using sub-optimal amounts of BAPTA, an intracellular Ca2+ chelator, which lowers lactate and rescues CD4+ T cell cycling. In addition, 7HF lowers T cell cycling with the combination of PMA and Ionomycin. However, 7HF increases CD4+ T cell cycling with sub-optimal activating signals: only PMA or anti-CD3. Furthermore, LPS-induced nitrite and IL6 production by peritoneal macrophages is inhibited by 7HF in a Ca2+-dependent manner. Studies with Ca2+ channel inhibitors, Ruthenium Red and 2-Aminoethoxydiphenyl borate, lowers the inhibitory effects of 7HF on CD4+ T cell and macrophage responses. In silico studies demonstrated that 7HF binds to Ca2+ channels, TRPV1, IP3R and SERCA, which is mechanistically important. Finally, intraperitoneal administration of 7HF lowers serum inflammatory cytokines, IFNγ and IL6, and reduces the effects of DSS-induced colitis with respect to colon length and colon damage. Overall, this study sheds mechanistic light on the anti-inflammatory potential of 7HF, a natural plant compound, in lowering immune responses.

Autoimmune-prone lpr mice exhibit a prolonged but lethal infection with an attenuated Salmonella Typhimurium strain.

Autoimmunity can potentially pre-dispose to, exacerbate or ameliorate pathogenic infections. The current study was designed to compare and understand the infection outcomes with Salmonella enterica serovar Typhimurium ATCC 14028s (S. Typhimurium) wild type (WT) and attenuated ΔrpoS strains, in autoimmune-prone lpr mice. C57BL/6 (B6) and B6/lpr (lpr) 6-8 weeks old mice were orally infected with S. Typhimurium WT and ΔrpoS strains. Disease outcomes were assessed with respect to survival, organ bacterial load, tissue damage and inflammation in infected mice. The acute infection stage (day 4) was examined and compared to the later stages (up to day 12) post ΔrpoS infection. S. Typhimurium WT exhibited an acute and lethal infection in both B6 and lpr mice. However, the ΔrpoS strain exhibited prolonged infection with reduced mortality in B6 mice but complete mortality in lpr mice. During late infection, bacterial load and serum IFNγ levels were higher in the ΔrpoS strain infected lpr mice compared to B6 mice. The ΔrpoS strain infected lpr mice also exhibited greater bacterial faecal shedding and greater tissue histopathological changes. Interestingly, ΔrpoS-infected B6 mice displayed minimal microbial load in the brain; however, sustained brain bacterial load was observed in ΔrpoS-infected lpr mice, corresponding to abnormal gait. Overall, S. Typhimurium ΔrpoS is competent in establishing infection but compromised in sustaining it. Nonetheless, lpr mice are less efficient in controlling this attenuated infection. The findings from the study demonstrate that genetic pre-disposition to autoimmunity is sufficient for greater host susceptibility to infection by attenuated S. Typhimurium strains.

The barley lectin, horcolin, binds high-mannose glycans in a multivalent fashion, enabling high-affinity, specific inhibition of cellular HIV infection.

N-Linked glycans are critical to the infection cycle of HIV, and most neutralizing antibodies target the high-mannose glycans found on the surface envelope glycoprotein-120 (gp120). Carbohydrate-binding proteins, particularly mannose-binding lectins, have also been shown to bind these glycans. Despite their therapeutic potency, their ability to cause lymphocyte proliferation limits their application. In this study, we report one such lectin named horcolin (Hordeum vulgare lectin), seen to lack mitogenicity owing to the divergence in the residues at its carbohydrate-binding sites, which makes it a promising candidate for exploration as an anti-HIV agent. Extensive isothermal titration calorimetry experiments reveal that the lectin was sensitive to the length and branching of mannooligosaccharides and thereby the total valency. Modeling and simulation studies demonstrate two distinct modes of binding, a monovalent binding to shorter saccharides and a bivalent mode for higher glycans, involving simultaneous interactions of multiple glycan arms with the primary carbohydrate-binding sites. This multivalent mode of binding was further strengthened by interactions of core mannosyl residues with a secondary conserved site on the protein, leading to an exponential increase in affinity. Finally, we confirmed the interaction of horcolin with recombinant gp120 and gp140 with high affinity and inhibition of HIV infection at nanomolar concentrations without mitogenicity.

T cell costimulation, checkpoint inhibitors and anti-tumor therapy.

The hallmarks of the adaptive immune response are specificity and memory. The cellular response is mediated by T cells which express cell surface T cell receptors (TCRs) that recognize peptide antigens in complex with major histocompatibility complex (MHC) molecules on antigen presenting cells (APCs). However, binding of cognate TCRs with MHC-peptide complexes alone (signal 1) does not trigger optimal T cell activation. In addition to signal 1, the binding of positive and negative costimulatory receptors to their ligands modulates T cell activation. This complex signaling network prevents aberrant activation of T cells. CD28 is the main positive costimulatory receptor on nai¨ve T cells; upon activation, CTLA4 is induced but reduces T cell activation. Further studies led to the identification of additional negative costimulatory receptors known as checkpoints, e.g. PD1. This review chronicles the basic studies in T cell costimulation that led to the discovery of checkpoint inhibitors, i.e. antibodies to negative costimulatory receptors (e.g. CTLA4 and PD1) which reduce tumor growth. This discovery has been recognized with the award of the 2018 Nobel prize in Physiology/Medicine. This review highlights the structural and functional roles of costimulatory receptors, the mechanisms by which checkpoint inhibitors work, the challenges encountered and future prospects.

Nitric oxide synthase 2 enhances the survival of mice during Salmonella Typhimurium infection-induced sepsis by increasing reactive oxygen species, inflammatory cytokines and recruitment of neutrophils to the peritoneal cavity.

Sepsis, a leading cause of death in intensive care units, is primarily caused due to an exaggerated immune response. The hyperactive inflammatory response mediated by immune cells against infectious organisms and their toxins results in host cell death and tissue damage, the hallmarks of septic shock. Therefore, molecules that modulate inflammatory responses are attractive therapeutic targets for sepsis. Nitric oxide (NO) is a signaling molecule, which is implicated in regulating diverse immune functions. Although, the protective roles of NO in infectious diseases are well documented, its importance in sepsis is controversial. In the present study, the effects of intra-peritoneal injection of mice with Salmonella Typhimurium, a Gram-negative intracellular pathogen, were studied which leads to a rapid upregulation of serum cytokines and infiltration of neutrophils to the peritoneal cavity. Surprisingly, the induction of inflammatory cytokines and chemokines, e.g. IL6 and CCL2, and the infiltration of neutrophils into the peritoneal cavity are mitigated in mice lacking Nitric oxide synthase 2 (NOS2). The reduced inflammatory response in Nos2-/- mice is accompanied by greater bacterial burden in the peritoneal cavity, lower thymic atrophy, higher liver damage and cardiovascular dysfunction followed by decreased survival. However, no significant differences are observed in other responses between C57BL/6 wild type (WT) and Nos2-/- mice: induction of glucocorticoids, phagocytic ability and apoptosis of peritoneal cells. This study clearly highlights the NOS2-dependent and -independent responses in this mouse model of peritonitis induced sepsis. Importantly, pre-treatment of Nos2-/- mice with DETA-NO, a NO donor, upon infection, restores neutrophil recruitment, reduces bacterial numbers in the peritoneal cavity, improves liver and cardio-vascular function and enhances survival. Interestingly, DETA-NO treatment does not significantly increase the survival of infected WT mice. The implications of these results and the complex roles of NO as a target molecule during sepsis are discussed.