Emerging druggable targets for immune checkpoint modulation in cancer immunotherapy: the iceberg lies beneath the surface.

The immune system serves as a fundamental defender against the initiation and progression of cancer. Failure of the immune system augments immunosuppressive action that leading to cancer manifestation. This immunosuppressive effect causes from significant alterations in immune checkpoint expression associated with tumoral progression. The tumor microenvironment promotes immune escape mechanisms that further amplifying immunosuppressive actions. Notably, substantial targeting of immune checkpoints has been pragmatic in the advancement of cancer research. This study highlights a comprehensive review of emerging druggable targets aimed at modulating immune checkpoint co-inhibitory as well as co-stimulatory molecules in response to immune system activation. This modulation has prompted to the development of newer therapeutic insights, eventually inducing immunogenic cell death through immunomodulatory actions. The study emphasizes the role of immune checkpoints in immunogenic regulation of cancer pathogenesis and explores potential therapeutic avenues in cancer immunotherapy.Modulation of Immunosuppressive and Immunostimulatory pathways of immune checkpoints in cancer immunotherapy.

Apple polyphenol phloretin complexed with ruthenium is capable of reprogramming the breast cancer microenvironment through modulation of PI3K/Akt/mTOR/VEGF pathways.

Our recent investigation directed to synthesize a novel ruthenium-phloretin complex accompanied by the study of antioxidant in addition to DNA binding capabilities, to determine the chemotherapeutic activity against breast carcinoma in vitro and in vivo. Ruthenium-phloretin complex was synthesized and characterized by different spectroscopic methods. The complex was further investigated to determine its efficacy in both MCF-7 and MDA-MB-231 human carcinoma cell lines and finally in an in vivo model of mammary carcinogenesis induced by DMBA in rats. Our studies confirm that the chelation of the metal and ligand was materialize by the 3-OH and 9-OH functional groups of the ligand and the complex is found crystalline and was capable of intercalating with CT-DNA. The complex was capable of reducing cellular propagation and initiate apoptotic events in MCF-7 and MDA-MB-231 breast carcinoma cell lines. Ruthenium-phloretin complex could modulate p53 intervene apoptosis in the breast carcinoma, initiated by the trail of intrinsic apoptosis facilitated through Bcl2 and Bax and at the same time down regulating the PI3K/Akt/mTOR pathway coupled with MMP9 regulated tumor invasive pathways. Ruthenium-phloretin chemotherapy could interrupt, revoke or suspend the succession of breast carcinoma by altering intrinsic apoptosis along with the anti-angiogenic pathway.

RNA transfer through tunneling nanotubes.

It was already suggested in the early '70's that RNA molecules might transfer between mammalian cells in culture. Yet, more direct evidence for RNA transfer in animal and plant cells was only provided decades later, as this field became established. In this mini-review, we will describe evidence for the transfer of different types of RNA between cells through tunneling nanotubes (TNTs). TNTs are long, yet thin, open-ended cellular protrusions that are structurally distinct from filopodia. TNTs connect cells and can transfer many types of cargo, including small molecules, proteins, vesicles, pathogens, and organelles. Recent work has shown that TNTs can also transfer mRNAs, viral RNAs and non-coding RNAs. Here, we will review the evidence for TNT-mediated RNA transfer, discuss the technical challenges in this field, and conjecture about the possible significance of this pathway in health and disease.

Differences in problem-solving between canid populations: Do domestication and lifetime experience affect persistence?

Past research has suggested that a variety of factors, phylogenetic and ontogenetic, play a role in how canines behave during problem-solving tasks and the degree to which the presence of a human influences their problem-solving behaviour. While comparisons between socialized wolves and domestic dogs have commonly been used to tease apart these predictive factors, in many cases a single dog population, often pets, have been used for these comparisons. Less is understood about how different populations of dogs may behave when compared with wolves, or with each other, during an independent problem-solving task. This experiment compared the independent persistence of four populations of canines (two groups of pet domestic dogs, a group of free-ranging domestic dogs, and human-socialized wolves) on an independent problem-solving task in the presence of an on looking human. Results showed that wolves persisted the most at the task while free-ranging dogs persisted the least. Free-ranging dogs gazed at the human experimenter for the longest durations during the task. While further research is needed to understand why these differences exist, this study demonstrates that dogs, even those living outside human homes as scavengers, show comparatively low levels of persistence when confronted with a solvable task in the presence of a human as well as significantly greater duration of human-directed gaze when compared with wolves.

Practice makes perfect: familiarity of task determines success in solvable tasks for free-ranging dogs (Canis lupus familiaris).

Domestic dogs' (Canis lupus familiaris) socio-cognitive faculties have made them highly sensitive to human social cues. While dogs often excel at understanding human communicative gestures, they perform comparatively poorly in problem-solving and physical reasoning tasks. This difference in their behaviour could be due to the lifestyle and intense socialization, where problem solving and physical cognition are less important than social cognition. Free-ranging dogs live in human-dominated environments, not under human supervision and are less socialized. Being scavengers, they often encounter challenges where problem solving is required in order to get access to food. We tested Indian street dogs in familiar and unfamiliar independent solvable tasks and quantified their persistence and dependence on a novel human experimenter, in addition to their success in solving a task. Our results indicate that free-ranging dogs succeeded and persisted more in the familiar task as compared to the unfamiliar one. They showed negligible amount of human dependence in the familiar task, but showed prolonged gazing and considerable begging behaviour to the human experimenter in the context of the unfamiliar task. Cognitive abilities of free-ranging dogs thus play a pivotal role in determining task-associated behaviours based on familiarity. In addition to that, these dogs inherently tend to socialize with and depend on humans, even if they are strangers. Our results also illustrate free-ranging dogs' low competence at physical cognitive tasks.

Potential role of immune cell therapy in gynecological cancer and future promises: a comprehensive review.

Gynecological malignancies are most leading causes of death among women worldwide. The high prevalence of gynecologic malignancies remains significant, necessitating to turn the novel treatment approach like immunotherapy, wherein cancer cells are killed by the invasion of immune system. In recent year, immunotherapy has mostly an advanced treatment approach to repressing the tumor cells survival, proliferation, and invasion via the activation of immune systems. Moreover, various types of immune cells including T-cells, B-cells, and dendritic cells are associated with the immunotherapeutic strategy in cancer treatment. Although the significant role of T-cells against cancer is well established, while B-cells and dendritic cells also play an important role against different gynecological cancer by regulating the immune system. This review focuses on that arena and highlight the role of immune cells in the treatment of gynaecological cancer. Various immune cell-based anticancer therapies such as T-cell therapies, Adoptive Cellular transfer, B-cell therapies as well as approaches to Dendritic Cell therapies have been discussed in detail. Furthermore, the clinical settings and future avenues regarding immunotherapy on gynecological cancer have also been reviewed and illuminated in the recent study.

Global analysis of contact-dependent human-to-mouse intercellular mRNA and lncRNA transfer in cell culture.

Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically,<1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

A Protocol for Non-biased Identification of RNAs Transferred Between Heterologous Mammalian Cell Types Using RNA Tagging, Cell Sorting, and Sequencing.

Intercellular communication is a major hallmark of multicellular organisms and is responsible for coordinating cell and tissue differentiation, immune responses, synaptic transmission, and both paracrine and endocrine signaling, for example. Small molecules, peptides, and proteins have all been studied extensively as mediators of intercellular communication; however, RNAs have also been shown recently to transfer between cells. In mammalian cells, microRNAs, tRNAs, short noncoding RNAs, mRNA fragments, as well as full-length mRNAs have all been shown to transfer between cells either by exosomes or by membrane nanotubes. We have previously described nanotube-mediated cell-cell transfer of specific mRNAs between heterologous mammalian cell types cultured in vitro. Here, we describe a simple method for the unbiased and quantitative identification of the complete range of transferred mRNAs (i.e., the mRNA transferome) in one population of mammalian cells following co-culture with another population. After co-culture, the individual cell populations are sorted by magnetic bead-mediated cell sorting and the transferred RNAs are then identified by downstream analysis methods, such as RNA sequencing. Application of this technique not only allows for determination of the mRNA transferome, but can also reveal changes in the native transcriptome of a cell population after co-culture. This can indicate the effect that co-culture and intercellular transfer of mRNA have upon cell physiology.

Transdermal Lipid Nanocarriers: A Potential Delivery System for Lornoxicam.

BACKGROUND: Lornoxicam, is a NSAID of the oxicam class. Its short duration of action owing to rapid elimination and gastrointestinal side effects limits its usefulness when administered orally. OBJECTIVE: The primary objective of the proposed work is to develop suitable lipid nanocarriers for transdermal delivery of Lornoxicam with increased drug residence time at local site of inflamation and in systemic circulation, overcoming undesired gastrointestinal side effects. METHOD: Lornoxicam loaded lipid nanocarriers like solid lipid nanocarriers (SLN), nano-structured lipid carriers (NLC) & nanoemulsions (NE) were prepared by high-speed homogenization technique. RESULT: The particle size, zeta potential, and polydispersity index as obtained, were in the range of 140- 193 nm, -22 to -32 mV, and 0.354-0.301 for SLN formulations and 146-201 nm, -23 to -30 mV, and 0.355-0.354 for NLC formulations respectively. Characterization of stable NE revealed that globule size, zeta potential and polydispersity index were within the range of 138 to 195 nm, -26.1±0.123 mV and 0.195 ± 1.231 respectively. It was also observed that entrapment efficacy and drug loading improved as the lipid concentration was increased. The results obtained from the in vitro permeation study and in vivo anti-inflammatory study showed controlled drug permeation, increased bioavailability, longer retention and better therapeutic potential of Lornoxicam after transdermal application of lipid nanoparticles as compared to conventional gel. CONCLUSION: It can be concluded that the developed lipid nanoparticle loaded gel was found to be a suitable drug delivery carrier for transdermal delivery of Lornoxicam to increase the residence time of drug in systemic circulation and to combat the gastrointestinal side effects.

In vitro & in vivo studies on lornoxicam loaded nanoemulsion gels for topical application.

The objective of this work was to increase the solubility, in vitro skin permeability of lornoxicam from semisolid topical formulations and also to investigate the in vivo potential of nanoemulsion formulation. Optimized lornoxicam loaded nanoemulsion was prepared successfully by spontaneous self-emulsification method and the size of the stable formulations was found within the range of 102 to 200 nm. The stable nanoemulsion formulations characterized for viscosity, droplet size, transmission electron microscopy (TEM) and refractive index. In vitro permeation rate of nanoemulsion and conventional gel of lornoxicam (LX) were determined. Prmeability parameters like steady-state flux (Jss), permeability coefficient (Kp), and enhancement ratio (Er) were significantly increased in nanoemulsion NE8 and the nanogel NG8 as compared to conventional gel (LG). In vivo studies revealed a significant increase in anti-inflammatory effects as compared with conventional gel of LX. The anti-inflammatory effects of formulation NG8 showed a significant increase in percent inhibition value when compared with control, this difference was found to be highly significant (p<0.001). This work shows for the first time that lornoxicam can be formulated into nanoemulsions and may show promise in enhancing solubility and permeation.

Solid lipid nanoparticles (SLNs) gels for topical delivery of aceclofenac in vitro and in vivo evaluation.

Solid lipid nanoparticles (SLN) are very potential formulations for topical delivery of anti-inflammatory and anti-arthritic drugs. The solid state of the lipid particles enable efficient drug encapsulation and controlled drug release. In the present study, the evaluation of different formulation parameters based on variation of concentration of lipid and cosurfactant was studied. The SLN gel formulations of the dispersions were compared to the SLN dispersions and with the marketed gel of aceclofenac. The SLNs were prepared by high speed homogenization and ultra-sonication method with fixed amount of aceclofenac (10%) and pluronic F68 (1.5%). The particle size, zeta potential and span of developed formulations was found to be within the range of 123 nm to 323 nm, -12.4 to -18.5 and 0.42 to 0.86 respectively as the lipid concentration was increased from 7.5% to 40%. The highest entrapment efficiency was found to be 75% with the formulation having lipid concentration of 30% and 0.85% of phospholipon 90G. Permeation rate and controlled release property of xanthan gum loaded SLN gel formulations and SLN dispersion was studied through excised pig skin for 24hr. The drug release of SLN gel formulations was better controlled as compare to SLN dispersions. In vivo anti-inflammatory study showed that action of aceclofenac was enhanced for SLN dispersion and gel formulations. The results indicated the superiority of SLN based formulations for topical delivery of aceclofenac.

Topical delivery of aceclofenac as nanoemulsion comprising excipients having optimum emulsification capabilities: preparation, characterization and in vivo evaluation.

OBJECTIVE: The aim of the present study was to investigate the potential of a nanoemulsion for topical delivery of aceclofenac using different excipients having optimum emulsifying ability rather than their solubilizing capacity. METHODS: The oil-in-water nanoemulsions were prepared by screening the excipients from the nanoemulsion region of pseudoternary phase diagram. The prepared nanoemulsions were subjected to different thermodynamic stability tests. The nanoemulsion formulations that passed thermodynamic stability tests were characterized for viscosity, droplet size, transmission electron microscopy, refractive index and in vitro skin permeation. The in vitro skin permeation profile of optimized nanoemulsion formulation (NE31, containing 23.85% Polyoxy-35-castor oil, 7.95% PEG 400 and 13.6% Triacetin) was compared with that of nanoemulsion gel (NG31) and marketed gel formulation (HIFENAC GEL (HIG)). In vivo anti-inflammatory efficacy studies were also carried out for NE31, NG31 and HIG. RESULTS: The significant (p < 0.001) increase in in vitro permeability and in vivo anti-inflammatory efficacy of the NG31 formulation was observed as compared with HIG formulation. CONCLUSION: It can be concluded that the selection of surfactant and cosurfactant on the basis of their emulsification capabilities other than the solubilizing capacity of drug is an important criterion for the formulation of nanoemulsion.

Solid lipid nanoparticles (SLNs) for topical delivery of aceclofenac by using xanthan gum: Ex vivo and in vivo evaluation.

Solid lipid nanoparticles (SLN) are very potential formulations for topical delivery of anti-inflammatory and anti-arthritic drugs. The solid state of the lipid particle enables efficient drug encapsulation and controlled drug release. In the present study the evaluation of different parameters based on variation of concentration of lipid and co-surfactant was studied. The SLN gel formulation of the same dispersion was compared with the SLN dispersion and with the marketed gel formulation of aceclofenac. At first the solid lipid nanoparticles were prepared by high speed homogenization and ultra-sonication method with fixed amount of aceclofenac (10%) and pluronic F68 (1.5%). The particle size, zeta potential and span was found to be within the range of 123 nm to 323 nm, -12.4 to -18.5 and 0.42 to 0.86 respectively as the lipid concentration was increasing from 7.5% to 40%. The highest entrapment efficiency was found to be 75% with the formulation having lipid concentration of 30% and 0.85% of phospholipon 90G (Phospholipid as co-surfactant). Particle surface morphology was measured by scanning electron microscope (SEM). Permeation rate and controlled release property of xanthan gum loaded SLN gel formulations and SLN dispersion was studied through the excised pig skin for 24hr and it was found that release rate of SLN gel formulation was more controlled as compare to SLN dispersions. In vivo pharmacodynamic study was carried out for 24 hr by injecting 0.1ml (w/v) CFA (Complete Fraud's Adjuvant). From the in vitro and in vivo study it was found that action of aceclofenac was enhanced by prepared SLN dispersion and gel formulations. The results indicated the utility of both SLN dispersion and SLN gel system for transdermal delivery of aceclofenac as excellent and logical.

Nanostructured Lipid Carriers (NLC)-Based Gel for the Topical Delivery of Aceclofenac: Preparation, Characterization, and In Vivo Evaluation.

The aim of this study was to prepare nanostructured lipid carriers (NLC)-based topical gel of aceclofenac for the treatment of inflammation and allied conditions. Stearic acid as the solid lipid, oleic acid as the liquid lipid, pluronic F68 as the surfactant, and phospholipon 90G as the co-surfactant were used. NLCs were prepared by melt-emulsification, low-temperature solidification, and high-speed homogenization methods. Characterization of the NLC dispersion was carried out through particle size analysis, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and an in vitro release study. The anti-inflammatory effect of the NLC gel was assessed by the rat paw edema technique and compared to marketed aceclofenac gel. The NLC dispersions exhibited d(90%) between 233 nm and 286 nm. All of the NLC showed high entrapment efficiency ranging from 67% to 82%. The particle size of NLC was further confirmed by the SEM study. The result of DSC showed that aceclofenac was dispersed in NLC in an amorphous state. Both the entrapment and release rate were affected by the percentage of oleic acid, but the method of preparation affected only the entrapment efficiency. The nanoparticulate dispersion was suitably gelled and assessed for in vitro permeation. Finally, NLC-based gels were found to possess superior (almost double) the anti-inflammatory activity compared to the marketed product. The anti-inflammatory activity of NLC gel showed a rapid onset of action, as well as a prolonged duration of action as compared with the marketed gel.

Evaluation of the role of erythropoietin and methotrexate in multiple sclerosis.

BACKGROUND: Erythropoietin, originally recognized for its role in erythropoiesis, has been shown to improve neurological outcome after stroke. Low-dose methotrexate is effective against certain inflammatory diseases, such as severe psoriasis and rheumatoid arthritis as well as Crohn's disease. Immunosuppressive effect of methotrexate also reduces the proportion of patients with chronic progressive multiple sclerosis with modest clinical benefits. Combination of erythropoietin and methotrexate can target neuroinflammation along with immunosupression. OBJECTIVE: To evaluate the role of erythropoietin and methotrexate in experimental autoimmune encephalomyelitis, a commonly used animal model of several degenerative human diseases like multiple sclerosis. MATERIALS AND METHODS: In the present study, C57BL/J6 mice were immunized with 200 µg of myelin basic protein (MBP) emulsified in complete Freund's adjuvant (CFA) supplemented with 1 mg/ml of killed mycobacterium tuberculosis (MBP: CFA in 1:1 ratio). These animals were given a combination of methotrexate and erythropoietin. Neurological function tests were scored daily by grading of clinical signs. Cerebral histopathology was performed to detect inflammatory infiltrates and demyelination. RESULTS: Treatment with erythropoietin and methotrexate significantly improved the neurological function recovery, reduced inflammatory infiltrates, and demyelination as compared to controls possibly by stimulating oligodendrogenesis and down-regulating proinflammatory infiltrates. CONCLUSION: The findings suggest an adjunctive use of methotrexate in demyelinating disease.