The ex vivo toll-like receptor 7 tolerance induction in donor lymphocytes prevents murine acute graft-versus-host disease.

BACKGROUND AIMS: Acute graft-versus-host disease (aGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation, mediated by alloreactive donor T cells. Toll-like receptors (TLRs), a family of conserved pattern-recognition receptors (PRRs), represent key players in donors' T-cell activation during aGVHD; however, a regulatory, tolerogenic role for certain TLRs has been recognized in a different context. We investigated whether the ex vivo-induced TLR-2,-4,-7 tolerance in donor cells could prevent alloreactivity in a mismatched transplantation model. METHODS: TLR-2,-4,-7 tolerance was induced in mouse splenocytes, after stimulation with low doses of corresponding ligands. Cellular and molecular changes of the TLR-tolerant splenocytes and purified T cells were assessed by immunophenotypic and gene expression analyses. Incidence of aGVHD was evaluated by the clinical score and survival as well as histopathology of target tissues. RESULTS: Only the R848-induced TLR7 tolerance prevented aGVHD. The TLR7 ligand-induced tolerance lasted for a critical post-transplant period and was associated with distinct cellular and molecular signatures characterized by induction of regulatory T cells, reduced alloreactivity and balanced regulation of inflammatory signaling and innate immune responses. The TLR7-tolerant T cells preserved the immunological memory and generated in vitro virus-specific T cells upon antigen stimulation. The anti-aGVHD tolerization effect was direct and specific to TLR7 and required the receptor-ligand interaction; TLR7-/- T cells isolated from B6 TLR7-/- mice presented a distinct gene expression profile but failed to prevent aGVHD. DISCUSSION: We propose an effective and clinically applicable ex vivo approach for aGVHD prevention through a transient and reversible immune reprogramming exerted by TLR7-tolerant donor lymphocytes.

Plerixafor+G-CSF-mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy.

Globin gene therapy requires abundant numbers of highly engraftable, autologous hematopoietic stem cells expressing curative levels of ß-globin on differentiation. In this study, CD34+ cells from 31 thalassemic patients mobilized with hydroxyurea+granulocyte colony-stimulating factor (G-CSF), G-CSF, Plerixafor, or Plerixafor+G-CSF were transduced with the TNS9.3.55 ß-globin lentivector and compared for transducibility and globin expression in vitro, as well as engraftment potential in a xenogeneic model after partial myeloablation. Transduction efficiency and vector copy number (VCN) averaged 48.4 ± 2.8% and 1.91 ± 0.04, respectively, whereas expression approximated the one-copy normal ß-globin output. Plerixafor+G-CSF cells produced the highest ß-globin expression/VCN. Long-term multilineage engraftment and persistent VCN and vector expression was encountered in all xenografted groups, with Plerixafor+G-CSF-mobilized cells achieving superior short-term engraftment rates, with similar numbers of CD34+ cells transplanted. Overall, Plerixafor+G-CSF not only allows high CD34+ cell yields but also provides increased ß-globin expression/VCN and enhanced early human chimerism under nonmyeloablative conditions, thus representing an optimal graft for thalassemia gene therapy.

Hematopoietic stem cells and liver regeneration: differentially acting hematopoietic stem cell mobilization agents reverse induced chronic liver injury.

Bone marrow (BM) could serve as a source of cells facilitating liver repopulation in case of hepatic damage. Currently available hematopoietic stem cell (HSC) mobilizing agents, were comparatively tested for healing potential in liver fibrosis. Carbon tetrachloride (CCl4)-injured mice previously reconstituted with Green Fluorescent Protein BM were mobilized with Granulocyte-Colony Stimulating Factor (G-CSF), Plerixafor or G-CSF+Plerixafor. Hepatic fibrosis, stellate cell activation and oval stem cell frequency were measured by Gomori and by immunohistochemistry for a-Smooth Muscle Actin and Cytokeratin-19, respectively. Angiogenesis was evaluated by ELISA and immunohistochemistry. Quantitative real-time PCR was used to determine the mRNA levels of liver Peroxisome Proliferator-Activated Receptor gamma (PPAR-γ), Interleukin-6 (IL-6) and Tumor Necrosis-alpha (TNFα). BM-derived cells were tracked by double immunofluorescence. The spontaneous migration of mobilized HSCs towards injured liver and its cytokine secretion profile was determined in transwell culture systems. Either single-agent mobilization or the combination of agents significantly ameliorated hepatic damage by decreasing fibrosis and restoring the abnormal vascular network in the liver of mobilized mice compared to CCl4-only mice. The degree of fibrosis reduction was similar among all mobilized mice despite that G-CSF+Plerixafor yielded significantly higher numbers of circulating HSCs over other agents. The liver homing potential of variously mobilized HSCs differed among the agents. An extended G-CSF treatment provided the highest anti-fibrotic effect over all tested modalities, induced by the proliferation of hepatic stem cells and decreased hepatic inflammation. Plerixafor-mobilized HSCs, despite their reduced liver homing potential, reversed fibrosis mainly by increasing hepatic PPAR-γ and VEGF expression. In all groups, BM-derived mature hepatocytes as well as liver-committed BM stem cells were detected only at low frequencies, further supporting the concept that alternative mechanisms rather than direct HSC effects regulate liver recovery. Overall, our data suggest that G-CSF, Plerixafor and G-CSF+Plerixafor act differentially during the wound healing process, ultimately providing a potent anti-fibrotic effect.

Mesenchymal stem cells are conditionally therapeutic in preclinical models of rheumatoid arthritis.

OBJECTIVE: The role of mesenchymal stem cells (MSC) in experimental arthritis is undoubtedly conflicting. This study explored the effect of bone marrow-derived MSC in previously untested and pathogenetically different models of rheumatoid arthritis (RA). METHODS: MSC were tested both in an induced (adjuvant-induced) and a spontaneous (K/BxN) arthritis model. Arthritis was assessed clinically and histologically. The proliferation of splenocytes and fibroblast-like synoviocytes (FLS) in the presence of MSC was measured by radioactivity incorporation. Toll-like receptor (TLR) expression was measured by real-time PCR. T-regulatory cell (Treg) frequency, T-cell apoptosis and cytokine secretion were monitored by flow cytometry. RESULTS: MSC, in vitro, strongly inhibited critical cell populations; splenocytes and FLS. In contrast, MSC proved ineffective in vivo, unless they were administered before disease onset, an effect implying that the inflammatory arthritic milieu potentially abrogates MSC immunomodulatory properties. In order to alleviate inflammation before MSC infusion, the authors administered, at arthritis onset, a short course with a proteasome inhibitor, bortezomib, whereas MSC were infused when established disease was expected. The bortezomib plus MSC group demonstrated a significantly decreased arthritis score over arthritic, MSC-only, bortezomib-only groups, also confirmed by histology and immunohistochemistry. The bortezomib plus MSC combination restored TLR expression and Treg frequency in blood and normalised FLS and splenocyte proliferation, apoptosis and cytokine secretion. CONCLUSION: MSC lose their immunomodulatory properties when infused in the inflammatory micromilieu of autoimmune arthritis. Conditioning of the recipient with bortezomib alters the disease microenvironment enabling MSC to modulate arthritis. Should milieu limitations also operate in human disease, this approach could serve as a strategy to treat RA by MSC.

Gene Therapy For Beta-Thalassemia: Updated Perspectives.

Allogeneic hematopoietic stem cell transplantation was until very recently, the only permanent curative option available for patients suffering from transfusion-dependent beta-thalassemia. Gene therapy, by autologous transplantation of genetically modified hematopoietic stem cells, currently represents a novel therapeutic promise, after many years of extensive preclinical research for the optimization of gene transfer protocols. Nowadays, clinical trials being held on a worldwide setting, have demonstrated that, by re-establishing effective hemoglobin production, patients may be rendered transfusion- and chelation-independent and evade the immunological complications that normally accompany allogeneic hematopoietic stem cell transplantation. The present review will offer a retrospective scope of the long way paved towards successful implementation of gene therapy for beta-thalassemia, and will pinpoint the latest strategies employed to increase globin expression that extend beyond the classic transgene addition perspective. A thorough search was performed using Pubmed in order to identify studies that provide a proof of principle on the aforementioned topic at a preclinical and clinical level. Inclusion criteria also regarded gene transfer technologies of the past two decades, as well as publications outlining the pitfalls that precluded earlier successful implementation of gene therapy for beta-thalassemia. Overall, after decades of research, that included both successes and pitfalls, the path towards a permanent, donor-irrespective cure for beta-thalassemia patients is steadily becoming a realistic approach.

Potential synergistic effect of phosphodiesterase inhibitors with chemotherapy in lung cancer.

Purpose: Lung cancer remains the leading cause of cancer-related deaths worldwide and novel therapeutic approaches targeting crucial pathways are urgently needed to improve its treatment. Differentiation-based therapeutics (Methylxanthines) and phosphodiesterase inhibitors (type 4 and 5), have been implicated in cancer treatment. Our objectives were to capture any potential anti-tumor effect of these drug combinations with chemotherapeutic agents in vitro. Methods: Theophylline as Methylxanthines, Roflumilast as phosphodiesterase type 4 (PDE4) inhibitor and Sildenafil as phosphodiesterase type 5 (PDE5) inhibitor are the drugs that we combined with the chemotherapeutic agents (Docetaxel, Cisplatin and Carboplatin) in vitro. Lung cancer cell lines (NCI-H1048-Small cell lung cancer-SCLC, A549- Non-small cell lung cancer-NSCLC) were purchased from ATCC LGC Standards. At indicated time-point, following 24h and 48h incubation, cell viability and apoptosis were measured with Annexin V staining by flow cytometry. Statistical analysis was performed by GraphPad Prism. Results: In SCLC, following 48h incubation, platinum combinations of carboplatin with roflumilast and sildenafil (p<0.001) and carboplatin with theophylline and sildenafil showed increased apoptosis when compared to carboplatin alone. Concerning the combinations of cisplatin, when combined with roflumilast, theophylline and sildenafil appeared with increased apoptosis of that alone (p<0.001, 24h and 48h incubation). In NSCLC, the 24h incubation was not enough to induce satisfactory apoptosis, except for the combination of cisplatin with roflumilast and theophylline (p<0.05) when compared to cisplatin alone. However, following 48h incubation, carboplatin plus sildenafil, carboplatin plus sildenafil, theophylline and roflumilast showed more cytotoxicity when compared to carboplatin alone (p<0.001). Docetaxel combinations showed no statistically significant results. Conclusion: The synergistic effect of PDE inhibitors with platinum-based agents has been demonstrated in lung cancer. Our suggestion is that these combinations could be used as additive and maintenance treatment in combination to antineoplastic agents in lung cancer patients.

Prospects of gene therapy for pulmonary diseases: progress and limitations.

BACKGROUND: Despite the proof of principle that gene therapy can cure various monogenic diseases, limited clinical progress has been noted for gene therapy of the respiratory system. Certain anatomic features of the lungs, along with the suboptimal gene delivery vehicles utilized up to now, have significantly delayed successful clinical practice. Thus, the need for additional improvements towards safety and efficacy of the procedure is indispensable. OBJECTIVE: The objective of this work was to review the progress and limitations of gene therapy in the treatment of lung disease with a focus on monogenic disease, chronic obstructive pulmonary disease and asthma and to present studies that provide a proof of principle that it works in different model systems and in patients. METHOD: A thorough search was performed on the aforementioned topic using Pubmed in order to identify relevant manuscripts. Several gene therapy studies for monogenic disorders affecting other organs or systems were also taken into consideration. RESULTS: A hundred and thirty one papers were included. Inclusion criteria regarded novel gene transfer technologies of the past decade, as well as publications outlining the pitfalls that precluded earlier successful implementation of gene therapy for pulmonary diseases. CONCLUSION: Current gene transfer protocols and vector design require additional amelioration. The rapidly evolving and much promising technology of CRISPR/Cas9 might possibly overcome the hurdles posed to date for effective implementation of gene therapy and become the basis for the onset of new clinical trials.