Leishmania donovani mitogen-activated protein kinases as a host-parasite interaction interface.

Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response.

Neutrophil generation from hematopoietic progenitor cells and induced pluripotent stem cells (iPSCs): potential applications.

Neutrophils are the most frequent immune cell type in peripheral blood, performing an essential role against pathogens. People with neutrophil deficiencies are susceptible to deadly infections, highlighting the importance of generating these cells in host immunity. Neutrophils can be generated from hematopoietic progenitor cells (HPCs) and embryonic stem cells (ESCs) using a cocktail of cytokines. In addition, induced pluripotent stem cells (iPSCs) can be differentiated into various functional cell types, including neutrophils. iPSCs can be derived from differentiated cells, such as skin and blood cells, by reprogramming them to a pluripotent state. Neutrophil generation from iPSCs involves a multistep process that can be performed through feeder cell-dependent and feeder cell-independent manners. Various cytokines and growth factors, in particular, stem cell facto, IL-3, thrombopoietin and granulocyte colony-stimulating factor (G-CSF), are used in both methods, especially, G-CSF which induces the final differentiation of neutrophils in the granulocyte lineage. iPSC-derived neutrophils have been used as a valuable tool for studying rare genetic disorders affecting neutrophils. The iPSC-derived neutrophils can also be used for disease modeling, infection research and drug discovery. However, several challenges must be overcome before iPSC-derived neutrophils can be used therapeutically in transplantation medicine. This review provides an overview of the commonly employed protocols for generating neutrophils from HPCs, ESCs and iPSCs and discusses the potential applications of the generated cells in research and medicine.

Interleukin-7 potentiates MAPK10-elicited host-protective vaccine against Leishmania donovani.

Leishmania donovani causes the potentially fatal disease visceral leishmaniasis for which neither a vaccine nor an adjuvant for human use exists. Although interleukin-7 (IL-7) is implicated in CD4+ T-cell response stabilization, its anti-leishmanial function is uncertain. Therefore, we examined whether IL-7 would potentiate the efficacy of Leishmania major-expressed MAPK10 (LmjMAPK10; M10)-elicited anti-leishmanial host-protective response. We observed that aligning with IL-7R expression, IL-7 increased IFN-γ-secreting TH1 cell but reduced IL-4-producing TH2 cells and production of IL-10 and TGF-ß effectuating anti-leishmanial functions in susceptible BALB/c mouse-derived macrophages. Co-culturing IL-7-pre-treated L. donovani-infected macrophages with L. donovani-infected BALB/c-derived T cells induced IFN-γ-dominated TH1 type anti-leishmanial function. IL-7 treatment of L. donovani-infected BALB/c mice significantly reduced splenic and hepatic parasite loads. Co-culturing CD4+ T cells from IL to 7-treated mice with L. donovani-infected macrophages reduced amastigote numbers suggesting IL-7-elicited host-protective effector T cells. Priming BALB/c with M10 + IL-7 reduced the splenic parasite burden more effectively than that was observed in M10-primed mice. An enhanced protection against L. donovani infection was accompanied by enhanced IL-12 and IFN-γ, but suppressed IL-10 and IL-4, response and host-protective TH1 and memory T cells. These results indicate IL-7-induced leishmanial antigen-specific memory T cell response that protects a susceptible host against L. donovani infection.

Ascorbic acid modulates immune responses through Jumonji-C domain containing histone demethylases and Ten eleven translocation (TET) methylcytosine dioxygenase.

Ascorbic acid is a redox regulator in many physiological processes. Besides its antioxidant activity, many intriguing functions of ascorbic acid in the expression of immunoregulatory genes have been suggested. Ascorbic acid acts as a co-factor for the Fe+2 -containing α-ketoglutarate-dependent Jumonji-C domain-containing histone demethylases (JHDM) and Ten eleven translocation (TET) methylcytosine dioxygenasemediated epigenetic modulation. By influencing JHDM and TET, ascorbic acid facilitates the differentiation of double negative (CD4- CD8- ) T cells to double positive (CD4+ CD8+ ) T cells and of T-helper cells to different effector subsets. Ascorbic acid modulates plasma cell differentiation and promotes early differentiation of hematopoietic stem cells (HSCs) to NK cells. These findings indicate that ascorbic acid plays a significant role in regulating both innate and adaptive immune cells, opening up new research areas in Immunonutrition. Being a water-soluble vitamin and a safe micro-nutrient, ascorbic acid can be used as an adjunct therapy for many disorders of the immune system.

Decoding the contextual duality of CD40 functions.

Previously, we established that as a function of its mode of interaction with its ligand or cellular conditions such as membrane lipids, preexisting signaling intermediates activation status, a transmembrane receptor, as represented here with CD40, can induce counteractive cellular responses. Using CD40-binding peptides, recombinant mutated CD40-ligands, and an agonistic antibody, we have established the functional duality of CD40. CD40 builds up two constitutionally different signalosomes on lipid raft and non-raft membrane domains initiating two different signaling pathways. Although this initial signaling may be modified by the pre-existing signaling conditions downstream and may be subjected to feed-forward or negative signaling effects, the initial CD40-CD40L interaction plays a crucial role in the functional outcome of CD40. Herein, we have reviewed the influence of interaction between the CD40-CD40L evoking the functional duality of CD40 contingent upon different physiological states of the cells.

Toll-like receptor 2 selectively modulates Ras isoforms expression in Leishmania major infection.

Leishmania infection of macrophages results in altered Ras isoforms expression and Toll-like receptor-2 (TLR2) expression and functions. Therefore, we examined whether TLR2 would selectively alter Ras isoforms' expression in macrophages. We observed that TLR2 ligands- Pam3CSK4, peptidoglycan (PGN), and FSL- selectively modulated the expression of Ras isoforms in BALB/c-derived elicited macrophages. Lentivirally-expressed TLR1-shRNA significantly reversed this Ras isoforms expression profile. TLR2-deficient L. major-infected macrophages and the lymph node cells from the L. major-infected mice showed similarly reversed Ras isoforms expression. Transfection of the macrophages with the siRNAs for the adaptors- Myeloid Differentiation factor 88 (MyD88) and Toll-Interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP)- or Interleukin-1 Receptor-Associated Kinases (IRAKs)- IRAK1 and IRAK4- significantly inhibited the L. major-induced down-regulation of K-Ras, and up-regulation of N-Ras and H-Ras, expression. The TLR1/TLR2-ligand Pam3CSK4 increased IL-10 and TGF-ß expression in macrophages. Pam3CSK4 upregulated N-Ras and H-Ras, but down-regulated K-Ras, expression in C57BL/6 wild-type, but not in IL-10-deficient, macrophages. IL-10 or TGF-ß signaling inhibition selectively regulated Ras isoforms expression. These observations indicate the specificity of the TLR2 regulation of Ras isoforms and their selective modulation by MyD88, TIRAP, and IRAKs, but not IL-10 or TGF-ß, signaling.

Leishmania donovani Induces Multiple Dynamic Responses in the Metabolome Associated with Amastigote Differentiation and Maturation Inside the Human Macrophage.

Leishmania donovani infection of macrophages drives profound changes in the metabolism of both the host macrophage and the parasite, which undergoes different phases of development culminating in replication and propagation. However, the dynamics of this parasite-macrophage cometabolome are poorly understood. In this study, a multiplatform metabolomics pipeline combining untargeted, high-resolution CE-TOF/MS and LC-QTOF/MS with targeted LC-QqQ/MS was followed to characterize the metabolome alterations induced in L. donovani-infected human monocyte-derived macrophages from different donors at 12, 36, and 72 h post-infection. The set of alterations known to occur during Leishmania infection of macrophages, substantially expanded in this investigation, characterized the dynamics of the glycerophospholipid, sphingolipid, purine, pentose phosphate, glycolytic, TCA, and amino acid metabolism. Our results showed that only citrulline, arginine, and glutamine exhibited constant trends across all studied infection time points, while most metabolite alterations underwent a partial recovery during amastigote maturation. We determined a major metabolite response pointing to an early induction of sphingomyelinase and phospholipase activities and correlated with amino acid depletion. These data represent a comprehensive overview of the metabolome alterations occurring during promastigote-to-amastigote differentiation and maturation of L. donovani inside macrophages that contributes to our understanding of the relationship between L. donovani pathogenesis and metabolic dysregulation.

Isoform-specific functions of Ras in T-cell development and differentiation.

Ras GTPases, well characterized for their role in oncogenesis, are the cells' molecular switches that signal to maintain immune homeostasis through cellular development, proliferation, differentiation, survival, and apoptosis. In the immune system, T cells are the central players that cause autoimmunity if dysregulated. Antigen-specific T-cell receptor (TCR) stimulation activates Ras-isoforms, which exhibit isoform-specific activator and effector requirements, functional specificities, and a selective role in T-cell development and differentiation. Recent studies show the role of Ras in T-cell-mediated autoimmune diseases; however, there is a scarcity of knowledge about the role of Ras in T-cell development and differentiation. To date, limited studies have demonstrated Ras activation in response to positive and negative selection signals and Ras isoform-specific signaling, including subcellular signaling, in immune cells. The knowledge of isoform-specific functions of Ras in T cells is essential, but still inadequate to develop the T-cell-targeted Ras isoform-specific treatment strategies for the diseases caused by altered Ras-isoform expression and activation in T cells. In this review, we discuss the role of Ras in T-cell development and differentiation, critically analyzing the isoform-specific functions.

CD40 induces selective routing of Ras isoforms to subcellular compartments.

Ras GTPases are central to cellular signaling and oncogenesis. The three loci of the Ras gene encode for four protein isoforms namely Harvey-Ras (H-Ras), Kirsten-Ras (K-Ras 4A and 4B), and Neuroblastoma-Ras (N-Ras) which share ~ 80% sequence similarity and used to be considered functionally redundant. The small molecule inhibitors of Ras lack specificity for the isoforms leading to widespread toxicity in Ras-targeted therapeutics. Ras isoforms' tissue-specific expression and selective association with carcinogenesis, embryonic development, and infection suggested their non-redundancy. We show that CD40, an antigen-presenting cell (APC)-expressed immune receptor, induces selective relocation of H-Ras, K-Ras, and N-Ras to the Plasma membrane (PM) lipid rafts, mitochondria, endoplasmic reticulum (ER), but not to the Golgi complex (GC). The two palmitoylated Ras isoforms-H-Ras and N-Ras-have a similar pattern of colocalization into the lipid-rich raft microdomain of the PM at early time points when compared to non-palmitoylated K-Ras (4B) with polylysine residues. CD40-induced trafficking of H-Ras and K-Ras to mitochondria and ER was found to be similar but different from that of N-Ras. Trafficking of all the Ras isoforms to the GC was independent of CD40 stimulation. The receptor-driven trafficking and spatial segregation of H-Ras, K-Ras, and N-Ras imply isoform-specific subcellular signaling platforms for the functional non-redundancy of Ras isoforms. PDB structures have been modified to illustrate various signaling proteins.

Evolvability and emergence of tumor heterogeneity as a space-time function.

The loss of control of cell proliferation, apoptosis regulation and contact inhibition leads to tumor development. While benign tumors are restricted to their primary space, i.e. where these tumors first originate, the metastatic tumors not only disseminate- facilitated by hypoxia-driven neovascularization- to distant secondary sites but also show substantial changes in metabolism, tissue architectures, gene expression profiles and immune phenotypes. All these alterations result in radio-, chemo- and immune-resistance rendering these metastatic tumor cells refractory to therapy. Since the beginning of the transformation, these factors- which influence each other- are incorporated to the developing and metastasizing tumor. As a result, the complexities in the heterogeneity of tumor progressively increase. This space-time function in the heterogeneity of tumors is generated by various conditions and factors at the genetic as well as microenvironmental levels, for example, endogenous retroviruses, methylation and epigenetic dysregulation that may be etiology-specific, cancer associated inflammation, remodeling of the extracellular matrix and mesenchymal cell shifted functions. On the one hand, these factors may cause de-differentiation of the tumor cells leading to cancer stem cells that contribute to radio-, chemo- and immune-resistance and recurrence of tumors. On the other hand, they may also enhance the heterogeneity under specific microenvironment-driven proliferation. In this editorial, we intend to underline the importance of heterogeneity in cancer progress, its evaluation and its use in correlation with the tumor evolution in a specific patient as a field of research for achieving precise patient-tailored treatments and amelioration of diagnostic (monitoring) tools and prognostic capacity.

Meta-analyses reveal serum or plasma Interleukin-6 as a biomarker for malignant ovarian neoplasia.

BACKGROUND: Interleukin-6 (IL-6) has been implicated in various malignancies, including ovarian cancer. However, mixed results have been observed regarding IL-6 levels in different ovarian conditions. This meta-analysis was performed to determine IL-6 levels in the peritoneal fluid and peripheral blood among patients with various adnexal masses. METHODS: Most popular English databases were searched using a predefined search formula. All studies comparing IL-6 levels in plasma, serum or peritoneal fluid of patients with benign tumors, ovarian neoplasms, and healthy controls were included based on inclusion and exclusion criteria. RESULTS: 5953 patients from 22 primary publications raging from 1994 to 2021 were included in the meta-analyses. A pooled IL-6 Mean Difference (MD) of 41 pg/mL for malignant tumors compared to benign ones, with a Confidence Interval (CI) between 19.8 and 62.2, a Z-score of 3.79, and statistical significance with a p = 0.0002 was observed. Pooled results for healthy versus benign ovarian conditions showed an MD of 5.45 pg/mL for serum or plasma IL-6 measurements in favor of benign tumors (CI:0.66-10.25, Z = 2.23 and p = 0.03). The analysis showed an MD for IL-6 levels of 19.59 pg/mL for healthy controls versus malignant ovarian tumors. Peritoneal fluid measurements regarding IL-6's levels showed no significant difference between benign or malignant masses. DISCUSSION/CONCLUSIONS: Higher levels of plasma or serum IL-6 in ovarian neoplasia patients compared to benign conditions or healthy controls identify IL-6 as a discerning factor between benign or malignant ovarian tumors and a potential biomarker for ovarian malignancy.

High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP.

Introduction: Non-Hodgkin Lymphoma (NHL) is a heterogeneous lymphoproliferative malignancy with B cell origin. Combinatorial treatment of rituximab, cyclophsphamide, hydroxydaunorubicin, oncovin, prednisone (R-CHOP) is the standard treatment regimen for NHL, yielding a complete remission (CR) rate of 40-50%. Unfortunately, considerable patients undergo relapse after CR or initial treatment, resulting in poor clinical implications. Patient's response to chemotherapy varies widely from static disease to cancer recurrence and later is primarily associated with the development of multi-drug resistance (MDR). The immunosuppressive cells within the tumor microenvironment (TME) have become a crucial target for improving the therapy efficacy. However, a better understanding of their involvement is needed for distinctive response of NHL patients after receiving chemotherapy to design more effective front-line treatment algorithms based on reliable predictive biomarkers. Methods: Peripheral blood from 61 CD20+ NHL patients before and after chemotherapy was utilized for immunophenotyping by flow-cytometry at different phases of treatment. In-vivo and in-vitro doxorubicin (Dox) resistance models were developed with murine Dalton's lymphoma and Jurkat/Raji cell-lines respectively and impact of responsible immune cells on generation of drug resistance was studied by RT-PCR, flow-cytometry and colorimetric assays. Gene silencing, ChIP and western blot were performed to explore the involved signaling pathways. Results: We observed a strong positive correlation between elevated level of CD33+CD11b+CD14+CD15- monocytic MDSCs (M-MDSC) and MDR in NHL relapse cohorts. We executed the role of M-MDSCs in fostering drug resistance phenomenon in doxorubicin-resistant cancer cells in both in-vitro, in-vivo models. Moreover, in-vitro supplementation of MDSCs in murine and human lymphoma culture augments early expression of MDR phenotypes than culture without MDSCs, correlated well with in-vitro drug efflux and tumor progression. We found that MDSC secreted cytokines IL-6, IL-10, IL-1ß are the dominant factors elevating MDR expression in cancer cells, neutralization of MDSC secreted IL-6, IL-10, IL-1ß reversed the MDR trait. Moreover, we identified MDSC secreted IL-6/IL-10/IL-1ß induced STAT1/STAT3/NF-κß signaling axis as a targeted cascade to promote early drug resistance in cancer cells. Conclusion: Our data suggests that screening patients for high titre of M-MDSCs might be considered as a new potential biomarker and treatment modality in overcoming chemo-resistance in NHL patients.

Bidirectional cytokine-microRNA control: A novel immunoregulatory framework in leishmaniasis.

As effector innate immune cells and as a host to the protozoan parasite Leishmania, macrophages play a dual role in antileishmanial immunoregulation. The 2 key players in this immunoregulation are the macrophage-expressed microRNAs (miRNAs) and the macrophage-secreted cytokines. miRNAs, as small noncoding RNAs, play vital roles in macrophage functions including cytokines and chemokines production. In the reverse direction, Leishmania-regulated cytokines alter miRNAs expression to regulate the antileishmanial functions of macrophages. The miRNA patterns vary with the time and stage of infection. The cytokine-regulated macrophage miRNAs not only help parasite elimination or persistence but also regulate cytokine production from macrophages. Based on these observations, we propose a novel immunoregulatory framework as a scientific rationale for antileishmanial therapy.

Leishmania intercepts IFN-γR signaling at multiple levels in macrophages.

IFN-γ, a type 2 interferon and a cytokine, is critical for both innate and adaptive immunity. IFN-γ binds to the IFN-γRs on the cell membrane of macrophages, signals through JAK1-STAT-1 pathway and induces IFN-γ-stimulated genes (ISGs). As Leishmania amastigotes reside and replicate within macrophages, IFN-γ mediated macrophage activation eventuate in Leishmania elimination. As befits the principle of parasitism, the impaired IFN-γ responsiveness in macrophages ensures Leishmania survival. IFN-γ responsiveness is a function of integrated molecular events at multiple levels in the cells that express IFN-γ receptors. In Leishmania-infected macrophages, reduced IFN-γRα expression, impaired IFN-γRα and IFN-γRß hetero-dimerization due to altered membrane lipid composition, reduced JAK-1 and STAT-1 phosphorylation but increased STAT-1 degradation and impaired ISGs induction collectively determine the IFN-γ responsiveness and the efficacy of IFN-γ induced antileishmanial function of macrophages. Therefore, parasite load is not only decided by the levels of IFN-γ produced but also by the IFN-γ responsiveness. Indeed, in Leishmania-infected patients, IFN-γ is produced but IFN-γ signalling is downregulated. However, the molecular mechanisms of IFN-γ responsiveness remain unclear. Therefore, we review the current understanding of IFN-γ responsiveness of Leishmania-infected macrophages.

Interactomic analysis reveals a homeostatic role for the HIV restriction factor TRIM5α in mitophagy.

The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.

TLR induced IL-27 plays host-protective role against B16BL6 melanoma in C57BL/6 mice.

Elicitation of the tumor-eliminating immune response is a major challenge, as macrophages- constituting a major component of solid tumor mass- play important roles in development, maintenance and tumor regression. The macrophage-expressed Toll-Like Receptors (TLRs) enhance macrophage function and their ability to activate T cells via secretion of cytokines, which may help in tumor regression. IL-27, a member of the IL-12 family of cytokines, is shown to exhibit anti-tumor and anti-angiogenic activities. Herein, we developed B16BL6 melanoma model in C75BL/6 mouse to dissect the crosstalk between TLRs and IL-27 in tumors. We report existence of a novel TLR- IL-27 feed-forward loop, whereby TLRs and IL-27 up-regulated each other's expression, which we found perturbed during melanoma tumorigenesis. Intra-tumoral injection of Imiquimod, a TLR7/8 ligand, reduced the tumor burden; the anti-tumor effect was reversed upon IL-27 and IL-27R silencing by intra-tumorally administered, lentivirally expressed IL-27 and IL-27R shRNA. The reduced tumor growth was accompanied by significantly fewer Treg cells but increased IFN-γ and granzyme B expression by CD8+ T cells. These data indicate the preventative role for TLR-induced IL-27 in aggressive and highly invasive melanoma.

Berberine mediates tumor cell death by skewing tumor-associated immunosuppressive macrophages to inflammatory macrophages.

BACKGROUND: Berberine is a plant-derived alkaloid with potent anti-cancer activities. Berberine may redirect the tumor-promoting immunosuppressive M2 macrophages, to tumoricidal activated M1 macrophages. But such an anti-tumor function remains to be demonstrated. HYPOTHESIS: Polarization of macrophages to an immunosuppressive phenotype within the tumor microenvironment promotes tumor growth and contributes to resistance to chemotherapy. We examined if berberine would target macrophage polarization to reinstate anti-tumor immune response. STUDY DESIGN: Using a B16F10 mouse melanoma model, we assessed berberine-induced re-polarization of immunosuppressive M2 macrophages to anti-tumor M1 macrophages and subsequent T-cell activation within the immunosuppressive tumor microenvironment. METHODS: The B16F10 culture supernatant along with tumor antigen was used as tumor mimicking conditioned medium (CM). The bone marrow-derived macrophages were cultured in CM for 5 days. The CM-induced skewing of macrophages to M2-like phenotype was confirmed by flow cytometry and ELISA. The T-cells were co-cultured with macrophages to decipher the effect of berberine on T-cell differentiation. In vivo efficacy of berberine was analyzed using melanoma model of solid tumor. RESULTS: Berberine inhibited rIL-6-induced STAT-3 phosphorylation and IL-10 release from B16F10 cells. It enhanced tumor antigen-induced IL-1ß, IL-12 and TNFα, but suppressed IL-6 and TGF-ß release. Berberine significantly prevented the tumor antigen-mediated IL-10-enhanced IL-6 and TGF-ß expression. The CM skewed the bone marrow-derived macrophages to CD206-high but MHC-II-low M2-like tumor-associated macrophages. Berberine partially prevented the generation of these macrophages and was associated with reduced C/EBPß and Egr2 mRNA expression and lowered IL-10 and TGF-ß production. Berberine significantly reduced Arginase-1 expression in CM-treated M1 and M2-like macrophages. Berberine increased MHC-II and CD40 expression on the macrophages augmenting the CTL activity and the number of IFNγ-producing CD4+ T-cells. Berberine significantly lowered tumor volume, weight and enhanced the frequency of M1-like macrophages in mice. CONCLUSION: These data indicate that berberine interferes with pro-tumor macrophage polarization and IL-10 and TGF-ß release but restores Tcell anti-tumor cytotoxicity in the tumor microenvironment.

Olive leaves extract alleviates inflammation and modifies the intrinsic apoptotic signal in the leukemic bone marrow.

Introduction: Current anti-leukemic chemotherapies with multiple targets suffer from side effects. Synthetic drugs with huge off-target effects are detrimental to leukemic patients. Therefore, natural plant-based products are being increasingly tested for new anti-leukemic therapy with fewer or no side effects. Herein, we report the effect of ethanolic olive leaves extract (EOLE) on the K562 cell line and on the bone marrow (BM) of N-ethyl-N-nitrosourea (ENU)-induced leukemic mice. Methods: Using standard methodologies, we assessed viability, chromatin condensation, and induction of apoptosis in EOLE-treated K562 cells in-vitro. The anti-leukemic activity of EOLE was assayed by measuring ROS, levels of various cytokines, expression of iNOS and COX-2 gene, and changes in the level of important apoptosis regulatory and cell signaling proteins in-vivo. Result: K562 cells underwent apoptotic induction after exposure to EOLE. In the BM of leukemic mice, EOLE therapy decreased the number of blast cells, ROS generation, and expression of NF-κB and ERK1/2. IL-6, IL-1ß, TNF-α, iNOS, and COX-2 were among the inflammatory molecules that were down-regulated by EOLE therapy. Additionally, it decreased the expression of anti-apoptotic proteins BCL2A1, BCL-xL, and MCL-1 in the BM of leukemic mice. Discussion: Chronic inflammation and anomalous apoptotic mechanism both critically contribute to the malignant transformation of cells. Inflammation in the tumor microenvironment promotes the growth, survival, and migration of cancer cells, accelerating the disease. The current investigation showed that EOLE treatment reduces inflammation and alters the expression of apoptosis regulatory protein in the BM of leukemic mice, which may halt the progression of the disease.

CD40L protects against mouse hepatitis virus-induced neuroinflammatory demyelination.

Neurotropic mouse hepatitis virus (MHV-A59/RSA59) infection in mice induces acute neuroinflammation due to direct neural cell dystrophy, which proceeds with demyelination with or without axonal loss, the pathological hallmarks of human neurological disease, Multiple sclerosis (MS). Recent studies in the RSA59-induced neuroinflammation model of MS showed a protective role of CNS-infiltrating CD4+ T cells compared to their pathogenic role in the autoimmune model. The current study further investigated the molecular nexus between CD4+ T cell-expressed CD40Ligand and microglia/macrophage-expressed CD40 using CD40L-/- mice. Results demonstrate CD40L expression in the CNS is modulated upon RSA59 infection. We show evidence that CD40L-/- mice are more susceptible to RSA59 induced disease due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS on day 10 p.i. Additionally, CD40L-/- mice exhibited severe demyelination mediated by phagocytic microglia/macrophages, axonal loss, and persistent poliomyelitis during chronic infection, indicating CD40-CD40L as host-protective against RSA59-induced demyelination. This suggests a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to immunosuppression which holds only for autoimmune mechanisms of inflammatory demyelination.

Ras Isoforms from Lab Benches to Lives-What Are We Missing and How Far Are We?

The central protein in the oncogenic circuitry is the Ras GTPase that has been under intense scrutiny for the last four decades. From its discovery as a viral oncogene and its non-oncogenic contribution to crucial cellular functioning, an elaborate genetic, structural, and functional map of Ras is being created for its therapeutic targeting. Despite decades of research, there still exist lacunae in our understanding of Ras. The complexity of the Ras functioning is further exemplified by the fact that the three canonical Ras genes encode for four protein isoforms (H-Ras, K-Ras4A, K-Ras4B, and N-Ras). Contrary to the initial assessment that the H-, K-, and N-Ras isoforms are functionally similar, emerging data are uncovering crucial differences between them. These Ras isoforms exhibit not only cell-type and context-dependent functions but also activator and effector specificities on activation by the same receptor. Preferential localization of H-, K-, and N-Ras in different microdomains of the plasma membrane and cellular organelles like Golgi, endoplasmic reticulum, mitochondria, and endosome adds a new dimension to isoform-specific signaling and diverse functions. Herein, we review isoform-specific properties of Ras GTPase and highlight the importance of considering these towards generating effective isoform-specific therapies in the future.