Osnat Almogi-Hazan

Preimplantation Factor (PIF*) reverses neuroinflammation while promoting neural repair in EAE model

INTRODUCTION Embryo-derived PIF modulates systemic maternal immunity without suppression. Synthetic analog (sPIF) prevents juvenile diabetes, preserves islet function, reducing oxidative stress/protein misfolding. We investigate sPIF effectiveness in controlling neuroinflammation/MS. METHODS Examine sPIF-induced protection against harsh, clinical-relevant murine EAE-PLP acute and chronic models. Evaluate clinical indices: circulating cytokines, spinal cord histology, genome, canonical global proteome, cultured PLP-activated splenocytes cytokines, and immunophenotype. RESULTS Short-term, low-dose sPIF prevented paralysis development and lowered mortality (P<0.05). Episodic sPIF reversed chronic paralysis (P<0.0001) completely in >50%, by day 82. Prevention model: 12days post-therapy, sPIF reduced circulating IL12 ten-fold and inflammatory cells access to spinal cord. Regression model: sPIF blocked PLP-induced IL17 and IL6 secretions. Long-term chronic model: sPIF reduced spinal cord pro-inflammatory cytokines/chemokines, (ALCAM, CF1, CCL8), apoptosis-promoters, inflammatory cells access (JAM3, OPA1), solute channels (ATPases), aberrant coagulation factors (Serpins), and pro-antigenic MOG. Canonical proteomic analysis demonstrated reduced oxidative phosphorylation, vesicle traffic, cytoskeleton remodeling involved in neuro-cytoskeleton breakdown (tubulins), associated with axon re-assembly by (MTAPs)/improved synaptic transmission. CONCLUSION sPIF--through coordinated central and systemic multi-targeted action--reverses neuroinflammation/MS and imparts significant neuroprotective effects up to total paralysis resolution. Clinical testing is warranted and planned.

Immune Regulation and Oxidative Stress Reduction by Preimplantation Factor following Syngeneic or Allogeneic Bone Marrow Transplantation

Bone marrow transplantation (BMT), a well-established treatment for hematological diseases, is frequently hampered by graft-versus-host disease (GVHD) and/or by infections due to delay in immune restoration. Prelmplantation Factor (PIF) is an embryo-derived peptide whose physiological function is to regulate local and systemic immunity and promote transplant acceptance. Synthetic PIF’s effectiveness to regulate immune response following BMT was herein examined in murine model. PIF administration reduced GVHD following allogenic BMT, decreased skin, liver, and colon inflammation and down regulated GVHD-associated gene expression in the liver. iNOS gene expression was reduced both in liver and colon. In syngeneic BMT, PIF administration reduced proinflammatory genes expression and promoted mice weight recovery up to two months after transplantation. PIF immune-regulatory effects were mediated via interaction with monocytes, resulting in decreased iNOS expression and NO secretion in-vitro. Overall, we demonstrate that by regulating immune response after BMT, PIF reduces inflammation and oxidative stress, leading to transplant success.

Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIFs effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIFs role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented.

PreImplantation factor (PIF*) regulates systemic immunity and targets protective regulatory and cytoskeleton proteins.

Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders.

PreImplantation factor (PIF) therapy provides comprehensive protection against radiation induced pathologies.

Acute Radiation Syndrome (ARS) may lead to cancer and death and has few effective countermeasures. Efficacy of synthetic PIF treatment was demonstrated in preclinical autoimmune and transplantation models. PIF protected against inflammation and mortality following lethal irradiation in allogeneic bone marrow transplant (BMT) model. Herein, we demonstrate that PIF imparts comprehensive local and systemic protection against lethal and sub-lethal ARS in murine models. PIF treatment 2 h after lethal irradiation led to 100% survival and global hematopoietic recovery at 2 weeks after therapy. At 24 h after irradiation PIF restored hematopoiesis in a semi-allogeneic BMT model. PIF-preconditioning provided improved long-term engraftment. The direct effect of PIF on bone marrow cells was also demonstrated in vitro: PIF promoted pre-B cell differentiation and increased immunoregulatory properties of BM-derived mesenchymal stromal cells. PIF treatment also improved hematopoietic recovery and reduced systemic inflammatory cytokine production after sub-lethal radiation exposure. Here, PIF also prevented colonic crypt and basal membrane damage coupled with reduced nitric oxide synthetase (iNOS) and increased (B7h1) expression. Global upper GI gene pathway analysis revealed PIFs involvement in protein-RNA interactions, mitochondrial oxidative pathways, and responses to cellular stress. Some effects may be attributed to PIFs influence on macrophage differentiation and function. PIF demonstrated a regulatory effect on irradiated macrophages and on classically activated M1 macrophages, reducing inflammatory gene expression (iNOS, Cox2), promoting protective (Arg1) gene expression and inducing pro-tolerance cytokine secretion. Notably, synthetic PIF is stable for long-term field use. Overall, clinical investigation of PIF for comprehensive ARS protection is warranted.

Ex Vivo Induced Regulatory Human/Murine Mesenchymal Stem Cells as Immune Modulators

Over the past decade there has been a growing interest in using mesenchymal stem cells (MSCs) as an immune‐regulatory agent for prevention and treatment of various immune disorders including graft‐versus‐host disease (GVHD), transplanted organ rejection, and autoimmune diseases. However, the high diversity in the results from clinical trials using MSCs for such disorders emphasizes the need for MSCs to be “professionalized” ex vivo to a more defined regulatory phenotype before administering to patients. To this aim, we have established an ex vivo immunomodulatory triple combination treatment (TCT) for MSCs, using IFNγ, TGFβ, and kynurenine. We show that pretreated MSCs acquire an immunomodulatory phenotype, have improved regulatory functions, and upregulate the expression of inducible nitric oxide synthase, indoleamine 2,3‐dioxygenase, cyclooxygenase‐2 (COX2), heme oxygenase 1, leukemia inhibitory factor (LIF), and programmed death ligand 1. We define the pathway of kynurenine induced aryl hydrocarbon receptor activation in MSCs and how it contributes to the upregulation of COX2 expression and IL‐6 downregulation. The combination of reduced IL‐6 secretion with enhanced LIF expression leads to the inhibition of Th17 differentiation in coculture of TCT MSCs and lymphocytes. To test the immunomodulatory function of TCT MSCs in vivo, we used the cells as GVHD prophylaxis in a GVHD mouse model. TCT MSCs administration significantly decreased GVHD score and improved mouse survival. Importantly, single administration could attenuate disease symptoms for more than 3 weeks. Based on these results, we suggest considering TCT MSCs as an improved cell therapy for systemic diseases with an underlying inflammatory and immunologic etiology. Stem Cells 2015;33:2256–2267

Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation

TCR‐mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ‐chain motifs, mediating the TCRs direct interaction with actin and inducing actin bundling. While T cells expressing the ζ‐chain mutated in these motifs lack cytoskeleton (actin) associated (cska)‐TCRs, they express normal levels of non‐cska and surface TCRs as cells expressing wild‐type ζ‐chain. However, such mutant cells are unable to display activation‐dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR‐actin linkage, providing the missing gap linking between TCR‐mediated Ag recognition, specific cytoskeleton orientation toward the T‐cell–APC interacting pole and long‐lived IS maintenance.

Failure of chimerism formation and tolerance induction from Fas ligand mutant bone marrow donors after nonmyeloablative conditioning

Formation of donor-recipient mixed chimerism after nonmyeloablative conditioning allows co-existence of donor and recipient hematopoietic stem cells, with solid organ allograft tolerance and less likeliness of graft versus host development. Using a post-transplant bronchiolitis obliterans murine model, we aimed to test the hypothesis that allograft preservation after mixed chimerism formation is dependent on the presence of a functional Fas ligand (FasL) on donor hematopoietic cells. To form mixed chimerism, two aliquots of 30 × 10(6) whole bone marrow cells (BMC) from either wild-type C57BL/6 in one group, or transgenic gld mice with mutant FasL (d = 0 and 2+) in the other were used, with both groups receiving intravenous busulfan (10mg/kg) on d-1 and intraperitoneal cyclophosphamide (200mg/kg) on d+1. Tracheal allografts obtained from C57BL/6 mice were implanted into recipient BALB/c mice subcutaneously on d = 0. Tracheal allografts were harvested at d+28 post-transplant and were evaluated by histopathology. Mixed chimerism formation was detected in wild type C57BL/6 whole BMC recipients, which was accompanied by tracheal allograft acceptance with near normal structure at d+28 post implantation. However, in recipients of FasL mutant whole BMC, neither mixed chimerism formation nor tracheal allograft acceptance was obtained. We thus conclude that bone marrow cells lacking functional FasL molecules could not be engrafted in allogeneic recipients to form stable mixed chimerism after nonmyeloablative conditioning, thus not allowing tracheal allograft acceptance.

The cytoskeleton-associated TCR ζ chain is constitutively phosphorylated in the absence of an active p56lck form

The TCR recognizes peptide‐MHC complexes and transmits activation signals leading to cellular responses. We have previously characterized two TCR populations expressed on the T cell surface; one is linked to the cytoskeleton via a detergent‐insoluble cytoskeleton‐associated ζ (cska‐ζ) chain, while the other is detergent soluble and not linked to the cytoskeleton. The cska‐ζ form displays unique properties: it is constitutively phosphorylated, does not undergo hyperphosphorylation upon TCR stimulation as opposed to its non‐cytoskeleton‐associated counterpart (non‐cska‐ζ) and it maintains a molecular mass of 16 kDa. It is well established that p56lck and possibly p59fyn are responsible for the generation of the 21 / 23‐kDa phosphorylated detergent‐soluble ζ form. We now demonstrate that the posphorylation of cska‐ζ does not require the activity of p56lck. We also show that although Lck does not phosphorylate cska‐ζ in vivo, it retains the capacity to phosphorylate cska‐ζ in vitro. Moreover, differences in ζ‐associated kinase activity were detected for non‐cska‐ζ and cska‐ζ. Our results indicating that different kinases phosphorylate the two ζ forms are consistent with a growing consensus that each TCR form may regulate distinct cellular functions.

In vitro Influence of Different Fatty Acids onthe Pharmacological Effect of Temozolomide

Introduction: Glioblastoma multiforme is the most malignant cancer in the human body with limited treatment options, in which Temozolomide (TMZ) is the conventional chemotherapeutic agent (CA) often prescribed for this condition. Fatty acids (FAs) per se, including polyunsaturated fatty acids (PUFAs), have been prescribed as adjuvants molecules for general cancer treatment. However, little is known about potential interactions between these lipids and the CAs. TMZ was combined with different FAs to access the nature of the interactions between these compounds. Methods: Two in vitro barrier models of the gut-brain axis (GBA) and the blood-brain barrier (BBB) were used. In each of them, 2-hydroxyoleic acid (2-OHOA); gamma-linolenic acid (GLA); and fish oil (FO) which contains both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were used as treatments. Results: In both GBA and BBB models, the combination of TMZ with each FA elicited antagonistic effects, interfering with the transepithelial electrical resistance (TEER) and producing less or no response in the inhibition of T98G cell proliferation. This was associated with alterations in cell morphology, β-catenin and E-cadherin protein expression, and induction of lipid droplets. Conclusion: Using our in vitro barrier models, we concluded that the FA might negatively interfere with TMZ`s cytotoxic effect. On the other hand, TMZ also may play a negative role in the antitumor action of specific FA like GLA or 2-OHOA, as observed in the GBA model. These results call into question the clinical utilization of these FA as nutritional adjuvants for patients under the use of TMZ.