Synthesis and characterization of two self-assembled [Cu6Gd3] and [Cu5Dy2] complexes exhibiting the magnetocaloric effect, slow relaxation of magnetization, and anticancer activity.

Two new paths for coordination driven self-assembly reactions under the binding support of 2-((1-hydroxy-2-methylpropan-2-ylimino)methyl)-6-methoxyphenol (H2L) have been discovered from the reactions of Cu(ClO4)2·6H2O, NEt3 and GdCl3/DyCl3·6H2O in MeOH/CHCl3 (2 : 1) medium. A similar synthetic protocol is useful to provide two different types of self-aggregated molecular clusters [Cu6Gd3(L)3(HL)3(µ3-Cl)3(µ3-OH)6(OH)2]ClO4·4H2O (1) and [Cu5Dy2(L)2(HL)2(µ-Cl)2(µ3-OH)4(ClO4)2(H2O)6](ClO4)2·2NHEt3Cl·21H2O (2). The adopted reaction procedure established the importance of the HO- and Cl- ions in the mineral-like growth of the complexes, derived from solvents and metal ion salts. In the case of complex 1, one GdIII center has been trapped at the central position of the core upheld by six µ3-OH and three µ3-Cl groups, whereas for complex 2 one CuII center was trapped using four µ3-hydroxo and two µ-chlorido groups. The magnetothermal behavior of 1 has been examined for a magnetocaloric effect of -ΔSm = 11.3 J kg-1 K-1 at 2 K for ΔH = 7 T, whereas the magnetic susceptibility measurements of 2 showed slow magnetic relaxation with Ueff = 15.8 K and τ0 = 9.8 × 10-7 s in zero external dc field. Cancer cell growth inhibition studies proved the potential of both the complexes with interestingly high activity for the Cu6Gd3 complex against human lung cancer cells. Both complexes 1 and 2 also exhibited DNA and human serum albumin (HSA) binding abilities in relation to the involved binding sites and thermodynamics.

Calcitriol and Retinoic acid antagonize each other to suppress the production of IL-9 by Th9 cells.

Distinct T helper cells, including Th9 cells help maintain homeostasis in the immune system. Vitamins play pivotal role in the immune system through many mechanisms, including regulating the differentiation of T helper cells. Calcitriol (1,25-dihydroxyvitamin D3) and retinoic acid possess hormone-like properties and are the bioactive metabolites of vitamin D and A, respectively, that signal through heterodimers containing the common retinoid X receptor. In contrast to individual treatment with the vitamins that significantly attenuates IL-9 production from Th9 cells, Th9 cells treated with both vitamins demonstrated IL-9 production similar to untreated Th9 cells. This is associated with reciprocal expression of PU.1 and Foxp3. While the recruitment of PU.1 was significantly impaired to the Il9 gene in the presence of calcitriol or retinoic acid in Th9 cells, addition of both vitamins together increased the recruitment of PU.1 to the Il9 gene. Calcitriol and retinoic acid together impaired the recruitment of HDAC1 to the Il9 gene without impacting Gcn5 recruitment. Importantly, retinoic acid negated the effect of calcitriol and impaired the binding of VDR on the Il9 gene by dampened VDR-RXR formation. Collectively, our data show that calcitriol and retinoic acid antagonize each other to regulate the differentiation of Th9 cells.

Calcitriol attenuates TLR2/IL-33 signaling pathway to repress Th9 cell differentiation and potentially limits the pathophysiology of rheumatoid arthritis.

There is limited information regarding the TLR2 signaling pathway involved in Th9 cell differentiation. The role of calcitriol in regulating TLR2-mediated Th9 cell development is unknown. Thus, we aimed to unravel the TLR2 signaling pathway in Th9 cells and its regulation by calcitriol. We have used n = 5-6 animals for each murine experiment. Human studies involved five healthy volunteers. Moreover, ten healthy individuals and ten RA patients were included in the study. Murine and human Th9 cells were treated with Calcitriol (100 nM) and Pam3CSK4 (2 µg/mL). The number of IL-9+ve cells was determined by flow cytometry. Real-time PCR was used to assess the gene expression. Serum 25(OH)D3 levels were determined by HPLC. We observed that TLR2 signals via IL-33/ST2 in Th9 cells. Increased TLR2 expression associated with increased IL9 expression and augmented disease severity in RA patients. Calcitriol attenuated TLR2 signaling in murine and human Th9 cells. Low serum vitamin D3 level negatively associated with increased IL-9 and TLR2 expression and disease severity in RA patients. Our data suggest a potential role of calcitriol to ameliorate the disease severity of RA patients.

Calcitriol Regulates the Differentiation of IL-9-Secreting Th9 Cells by Modulating the Transcription Factor PU.1.

Vitamin D can modulate the innate and adaptive immune system. Vitamin D deficiency has been associated with various autoimmune diseases. Th9 cells are implicated in the pathogenesis of numerous autoimmune diseases. Thus, we investigated the role of calcitriol (active metabolite of vitamin D) in the regulation of Th9 cell differentiation. In this study, we have unraveled the molecular mechanisms of calcitriol-mediated regulation of Th9 cell differentiation. Calcitriol significantly diminished IL-9 secretion from murine Th9 cells associated with downregulated expression of the Th9-associated transcription factor, PU.1. Ectopic expression of VDR in Th9 cells attenuated the percentage of IL-9-secreting cells. VDR associated with PU.1 in Th9 cells. Using a series of mutations, we were able to dissect the VDR domain involved in the regulation of the Il9 gene. The VDR-PU.1 interaction prevented the accessibility of PU.1 to the Il9 gene promoter, thereby restricting its expression. However, the expression of Foxp3, regulatory T cell-specific transcription factor, was enhanced in the presence of calcitriol in Th9 cells. When Th9 cells are treated with both calcitriol and trichostatin A (histone deacetylase inhibitor), the level of IL-9 reached to the level of wild-type untreated Th9 cells. Calcitriol attenuated specific histone acetylation at the Il9 gene. In contrast, calcitriol enhanced the recruitment of the histone modifier HDAC1 at the Il9 gene promoter. In summary, we have identified that calcitriol blocked the access of PU.1 to the Il9 gene by reducing its expression and associating with it as well as regulated the chromatin of the Il9 gene to regulate expression.

Rheumatoid arthritis: 'melting pot' of T helper subsets.

Rheumatoid arthritis (RA) is an autoimmune disorder that affects joints associated with inflammation leading to poor quality of life. The phenotype of RA is distinct from osteoarthritis (OA), the degenerative joint disorder. The annual incidence of RA is approximately 4 in 10,000 individuals. Studies suggest dysregulated T cell activation in the initiation and progression of RA. Distinct RA-associated allelic variants encode molecules involved in T-cell activation pathways. Additionally, RA is also associated with aberrant regulation and function of T helper cells. The interplay of distinct T helper cell subsets adds complexity to the regulation of RA. In this review we have aimed to understand the currently known biology of different Th subsets in the context of an autoimmune disease like rheumatoid arthritis and find potential therapeutic approaches to tackle the disease through modulation of responsible T cells.

Size-dependent cellular uptake and TLR4 attenuation by gold nanoparticles in lung adenocarcinoma cells.

AIM: To elucidate uptake mechanisms and immunomodulatory potential of differently sized gold nanoparticles (GNPs) in lung adenocarcinoma cells (A549) to enable their use as an adjunct therapy for treating inflammation-linked lung cancer. METHODS: Internalization of the synthesized (5, 15 and 30 nm) GNPs by various endocytosis pathways was determined. Immunomodulatory mechanisms induced by differently sized GNPs in A549 cells in the presence of TLR4 and TLR9 ligands were evaluated. RESULTS: GNPs were size-dependently internalized efficiently by A549 cells. Various sized GNPs downregulated the expression of proinflammatory signaling molecules (5 nm most potent). Mechanistically, 5-nm GNPs attenuated TLR4 signaling by downregulating TLR4 expression in A549 cells. CONCLUSION: Our study suggests the use of immunomodulatory GNPs as an adjunct therapy against inflammation-linked lung cancer.

A Decade of Th9 Cells: Role of Th9 Cells in Inflammatory Bowel Disease.

T helper cell subsets play a critical role in providing protection against offending pathogens by secreting specific cytokines. However, unrestrained T helper cell responses can promote chronic inflammation-mediated inflammatory diseases. Dysregulated T helper cell responses have been suggested to be involved in the pathogenesis of multiple inflammatory diseases, including allergic airway inflammation, rheumatoid arthritis, and inflammatory bowel disease (IBD) among others. Aberrant pro-inflammatory responses induced by Th1, Th2, and Th17 subsets are known to trigger IBD. IBD is a chronic inflammatory disease characterized by weight loss, diarrhea, pain, fever, and rectal bleeding. It poses a major health burden worldwide owing to the increased risk of colorectal cancer development. Despite numerous therapeutic advancements, IBD still remains a major health burden due to the inefficiency of the conventional therapies. Recently, IL-9-secreting Th9 cells are known to be involved in the pathogenesis of IBD. However, the role of Th9 cells and their secretory cytokine IL-9 in IBD is unclear. The functional relevance of Th9 cells is also relatively understudied in IBD. Thus, investigating the actual role of various T helper cell subsets including Th9 cells in IBD is essential to develop novel therapies to treat IBD. Here, we highlight the role of Th9 cells in promoting IBD. We discuss the mechanisms that might be employed by Th9 cells and IL-9 in promoting IBD and thereby propose potential targets for the treatment of Th9 cell-mediated IBD.

Striking the right immunological balance prevents progression of tuberculosis.

INTRODUCTION: Tuberculosis (TB) caused by infection with Mycobacterium tuberculosis (Mtb) is a major burden for human health worldwide. Current standard treatments for TB require prolonged administration of antimycobacterial drugs leading to exaggerated inflammation and tissue damage. This can result in the reactivation of latent TB culminating in TB progression. Thus, there is an unmet need to develop therapies that would shorten the duration of anti-TB treatment and to induce optimal protective immune responses to control the spread of mycobacterial infection with minimal lung pathology. FINDINGS: Granulomata is the hallmark structure formed by the organized accumulation of immune cells including macrophages, natural killer cells, dendritic cells, neutrophils, T cells, and B cells to the site of Mtb infection. It safeguards the host by containing Mtb in latent form. However, granulomata can undergo caseation and contribute to the reactivation of latent TB, if the immune responses developed to fight mycobacterial infection are not properly controlled. Thus, an optimal balance between innate and adaptive immune cells might play a vital role in containing mycobacteria in latent form for prolonged periods and prevent the spread of Mtb infection from one individual to another. CONCLUSION: Optimal and well-regulated immune responses against Mycobacterium tuberculosis may help to prevent the reactivation of latent TB. Moreover, therapies targeting balanced immune responses could help to improve treatment outcomes among latently infected TB patients and thereby limit the dissemination of mycobacterial infection.

Paradoxical activation of T cells via augmented ERK signaling mediated by a RAF inhibitor.

RAF inhibitors selectively block ERK signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T cell activation, based upon the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T cell activation and signaling in vitro and in vivo. We observe that T cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with CTLA-4 blockade in vivo potentiates T cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Lastly, this combination demonstrates superior anti-tumor activity, compared to either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T cell function by potentiating T cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models.

Safety and immunogenicity of a human and mouse gp100 DNA vaccine in a phase I trial of patients with melanoma.

A differentiation antigen commonly expressed on melanoma cells, gp100 is the target of infiltrating T cells. We conducted a phase I randomized cross-over trial of melanoma patients with either xenogeneic (mouse) or human gp100 plasmid DNA injected intramuscularly at three dosages (100, 500 or 1,500 microg) every three weeks for three doses. After the first three injections, patients were then immunized three times with gp100 from the other species. Peripheral blood samples were analyzed at various time points following 10-day culture with gp100 peptides using multi-parametric flow cytometry. A total of 19 patients were enrolled, with 18 assessable for immune function and survival. 14 (74%) were male, with a median age of 56 years (range, 20-82). All patients had no evidence of disease; 10 (53%) had stage III disease, 3 each (16%) had stage IIB and IV disease, 2 (11%) had choroidal and 1 (5%) had anal mucosal involvement. With a median follow-up of 30 months, median progression-free survival (PFS) is 44 months. Median survival is not reached. There was no grade 3/4 toxicity; the most common grade 1/2 toxicity was an injection site reaction in 12 patients (63%, all grade 1). Five patients developed CD8+ cells binding gp100(280-288) HLA-A2-restricted tetramer. One patient had an increase in CD8+ IFN-gamma+ cells. This xenogeneic immunization strategy was safe and associated with minimal toxicity. There was also evidence of immune response.

Biological systems of the host cell involved in Agrobacterium infection.

Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, represents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell-cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity--thought to participate in the Agrobacterium-mediated genetic transformation.

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.

pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants.

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation.