Glycopolymer Inhibitors of Galectin-3 Suppress the Markers of Tissue Remodeling in Pulmonary Hypertension.

Pulmonary hypertension is a cardiovascular disease with a low survival rate. The protein galectin-3 (Gal-3) binding ß-galactosides of cellular glycoproteins plays an important role in the onset and development of this disease. Carbohydrate-based drugs that target Gal-3 represent a new therapeutic strategy in the treatment of pulmonary hypertension. Here, we present the synthesis of novel hydrophilic glycopolymer inhibitors of Gal-3 based on a polyoxazoline chain decorated with carbohydrate ligands. Biolayer interferometry revealed a high binding affinity of these glycopolymers to Gal-3 in the subnanomolar range. In the cell cultures of cardiac fibroblasts and pulmonary artery smooth muscle cells, the most potent glycopolymer 18 (Lac-high) caused a decrease in the expression of markers of tissue remodeling in pulmonary hypertension. The glycopolymers were shown to penetrate into the cells. In a biodistribution and pharmacokinetics study in rats, the glycopolymers accumulated in heart and lung tissues, which are most affected by pulmonary hypertension.

Thymoma - disease of many faces.

A thymoma belongs to rather rare tumors, although it holds the first place among primary mediastinal tumors in adults. The biological characteristics of thymomas are considerably divergent. The tumors are usually asymptomatic at the begining, later due to a local spread, symptoms from the infiltration or compression of adjacent structures arise, they metastasize rarely. Thymomas are often associated with various autoimmune or endocrinologic diseases. The authors present case of the patient with less common paraneoplasia - autoimmune hemolytic anemia (AIHA). Main strategy of treatment thymom with AIHA isn´t clear. The most important seems to be the early started corticotherapy. Achieving remission of secondary AIHA has importace in following therapy of thymoma - surgical resection or chemotherapy. Thymectomy has positive effect on the course of associated paraneoplastic syndromes and prognosis in general.

Day 3 Poly (I:C)-activated dendritic cells generated in CellGro for use in cancer immunotherapy trials are fully comparable to standard Day 5 DCs.

BACKGROUND: Dendritic cells (DCs) are professional antigen-presenting cells that are capable of inducing immune responses. DC-based vaccines are normally generated using a standard 5- to 7-day protocol. To shorten the DC-based vaccine production for use in cancer immunotherapy, we have developed a fast DC protocol by comparing standard DCs (Day 5 DCs) and fast DCs (Day 3 DCs). METHODS: We tested the generation of Day 5 versus Day 3 DCs using CellGro media and subsequent activation by two activation stimuli: Poly (I:C) and LPS. We evaluated DC morphology, viability, phagocyte activity, cytokine production and ability to stimulate antigen-specific T cells. RESULTS: Day 5 and Day 3 DCs exhibited similar phagocytic capacity. Poly (I:C)-activated Day 5 DCs expressed higher levels of the costimulatory and surface molecules CD80, CD86 and HLA-DR compared to Poly (I:C)-activated Day 3 DCs. Nevertheless, LPS-activated Day 5 and Day 3 DCs were phenotypically similar. Cytokine production was generally stronger when LPS was used as the maturation stimulus, and there were no significant differences between Day 5 and Day 3 DCs. Importantly, Day 5 and Day 3 DCs were able to generate comparable numbers of antigen-specific CD8(+) T cells. The number of Tregs induced by Day 5 and Day 3 DCs was also comparable. CONCLUSION: We identified monocyte-derived DCs generated in CellGro for 3 days and activated using Poly (I:C) similarly potent in most functional aspects as DCs produced by the standard 5 day protocol. These results provide the rationale for the evaluation of faster protocols for DC generation in clinical trials.

Tracking the extramedullary PML-RARα-positive cell reservoirs in a preclinical model: biomarker of long-term drug efficacy.

Using an acute promyelocytic leukemia (APL) preclinical model, we show that oncogene-specific PCR (Polymerase Chain Reaction)-based assays allow to evaluate the efficacy of immunotherapy combining all-trans retinoic acid (ATRA) and a DNA-based vaccine targeting the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) oncogene. Kaplan-Meier survival analysis according to the peripheral blood PML-RARα normalized copy number (NCN) clearly shows that ATRA + DNA-treated mice with an NCN lower than 10 (43%) formed the group with a highly significant (p < 0.0001) survival advantage. Furthermore, a PCR assay was used to assess various tissues and organs for the presence of PML-RARα-positive cells in long-term survivors (n = 15). As expected, the majority of mice (n = 10) had no measurable tissue level of PML-RARα. However, five mice showed a weak positive signal in both the brain and spleen (n = 2), in the brain only (n = 2) and in the spleen only (n = 1). Thus tracking the oncogene-positive cells in long-term survivors reveals for the first time that extramedullary PML-RARα-positive cell reservoirs such as the brain may persist and be involved in relapses.

Unusual regression of severe recurrent lymphatic malformation of a face after contraception and pregnancy.

We report the case of a female who had suffered from progressive lymphatic malformation in the orbito-temporal region since childhood. Many surgical interventions were performed, including radical excision and shunt drainage. Despite aggressive surgical treatment, recurrence was observed after every intervention. Eventually, the condition regressed after the patient began taking a contraceptive. Moreover, it virtually disappeared after pregnancy.

IL-12 inhibits the TGF-ß-dependent T cell developmental programs and skews the TGF-ß-induced differentiation into a Th1-like direction.

The development and differentiation of T helper (Th) cell subsets is a highly plastic process which is strictly regulated by cytokines. Here we show that the transforming growth factor ß (TGF-ß)-dependent differentiation programs are negatively regulated by interleukin-12 (IL-12). The development of TGF-ß-induced regulatory T cells (iTregs) or TGF-ß/IL-6 activated Th17 cells from purified mouse CD4(+)CD25(-) T cells, stimulated with monoclonal antibody anti-CD3, was abrogated in the presence of IL-12 and a different developmental program was established. On the molecular level, IL-12 inhibited the expression of the lineage specific transcription factors Foxp3 and RORγt in developing Tregs and Th17 cells, respectively. Moreover, IL-12 was able to alter the development of iTregs and Th17 cells even when added to the differentiating cells after 48h of the culture. The cells activated in the presence of TGF-ß and IL-12 had an increased expression of the Th1 transcription factor T-bet, produced Th1 cytokines interferon γ and IL-2 and expressed IL-18 receptor and C-C chemokine receptor type 5 which are the phenotypic markers characteristic for Th1 cells. Furthermore, the cells activated in the presence of both TGF-ß and IL-12, and not of TGF-ß only, stimulated macrophages to produce nitric oxide. Altogether, these results indicate that IL-12 is a superior cytokine that has the ability to skew the already ongoing TGF-ß-dependent iTreg or Th17 developmental program into Th1-like direction.

The role of mouse mesenchymal stem cells in differentiation of naive T-cells into anti-inflammatory regulatory T-cell or proinflammatory helper T-cell 17 population.

Bone marrow-derived mesenchymal stem cells (MSCs) modulate immune response and can produce significant levels of transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6). These 2 cytokines represent the key factors that reciprocally regulate the development and polarization of naive T-cells into regulatory T-cell (Treg) population or proinflammatory T helper 17 (Th17) cells. In the present study we demonstrate that MSCs and their products effectively regulate expression of transcription factors Foxp3 and RORγt and control the development of Tregs and Th17 cells in a population of alloantigen-activated mouse spleen cells or purified CD4(+)CD25(-) T-cells. The immunomodulatory effects of MSCs were more pronounced when these cells were stimulated to secrete TGF-ß alone or TGF-ß together with IL-6. Unstimulated MSCs produce TGF-ß, but not IL-6, and the production of TGF-ß can be further enhanced by the anti-inflammatory cytokines IL-10 or TGF-ß. In the presence of proinflammatory cytokines, MSCs secrete significant levels of IL-6, in addition to a spontaneous production of TGF-ß. MSCs producing TGF-ß induced preferentially expression of Foxp3 and activation of Tregs, whereas MSC supernatants containing TGF-ß together with IL-6 supported RORγt expression and development of Th17 cells. The effects of MSC supernatants were blocked by the inclusion of neutralization monoclonal antibody anti-TGF-ß or anti-IL-6 into the culture system. The results showed that MSCs represent important players that reciprocally regulate the development and differentiation of uncommitted naive T-cells into anti-inflammatory Foxp3(+) Tregs or proinflammatory RORγt(+) Th17 cell population and thereby can modulate autoimmune, immunopathological, and transplantation reactions.

Graft survival and cytokine production profile after limbal transplantation in the experimental mouse model.

Limbal transplantation or limbal stem cell (LSC) transfer represents the only way to treat severe ocular surface damage or LSC deficiency. However, limbal allografts are promptly rejected in spite of extensive immunosuppressive therapy. To characterize immune response after limbal transplantation, we established an experimental model of limbal transplantation in the mouse. Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were grafted orthotopically in BALB/c mice and graft survival was evaluated. The presence of graft donor cells and the expression of IL-2, IL-4, IL-10, IFN-γ and inducible nitric oxide synthase (iNOS) mRNA in the grafts were detected by real-time PCR. While syngeneic grafts survived permanently, allografts were rejected in 9.0±1.8 days and xenografts in 6.5±1.1 days. The manifestation of clinical symptoms of rejection correlated with the disappearance of donor cells in the graft and in the recipient cornea. Intragraft expression of iNOS mRNA and distinct expression patterns of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines were detected during rejection of limbal allografts and xenografts. The limbal graft rejection was prevented with anti-CD4, but not anti-CD8 monoclonal antibody therapy. The results indicate that limbal grafts do not enjoy immune privilege of the eye and are promptly rejected by Th1 (allografts) or by a combined Th1 and Th2 (xenografts) type of immune response involving CD4+ cells and iNOS expression. Targeting this pathway may be an effective way to prevent and treat limbal graft rejection.

Poly I: C-activated dendritic cells that were generated in CellGro for use in cancer immunotherapy trials.

BACKGROUND: For clinical applications, dendritic cells (DCs) need to be generated using GMP-approved reagents. In this study, we tested the characteristics of DCs generated in two clinical grade culture media and activated by three maturation stimuli, Poly I: C, LPS and the mixture of proinflammatory cytokines in order to identify the optimal combination of culture media and activation stimulus for the clinical use. METHOD: We tested DCs generation using two GMP-certified culture media, CellGro and RPMI+5% human AB serum and evaluated DCs morphology, viability and capapability to mature. We tested three maturation stimuli, PolyI:C, LPS and the mixture of proinflammatory cytokines consisting of IL-1, IL-6, TNF and prostaglandin E2. We evaluated the capacity of activated DCs to induce antigen-specific T cells and regulatory T lymphocytes. RESULTS: Cell culture in CellGro resulted in a higher yield of immature DCs resulting from increased number of adherent monocytes. DCs that were generated in CellGro and activated using Poly I:C were the most efficient in expanding antigen-specific T cells compared to the DCs that were generated in other media and activated using LPS or the cocktail of proinflammatory cytokines. A comparison of all tested combinations revealed that DCs that were generated in CellGro and activated using Poly I:C induced low numbers of regulatory T cells. CONCLUSION: In this study, we identified monocyte-derived DCs that were generated in CellGro and activated using Poly I:C as the most potent clinical-grade DCs for the induction of antigen-specific T cells.

Treatment of ocular surface injuries by limbal and mesenchymal stem cells growing on nanofiber scaffolds.

Stem cell (SC) therapy represents a promising approach to treat a wide variety of injuries, inherited diseases, or acquired SC deficiencies. One of the major problems associated with SC therapy remains the absence of a suitable matrix for SC growth and transfer. We describe here the growth and metabolic characteristics of mouse limbal stem cells (LSCs) and mesenchymal stem cells (MSCs) growing on 3D nanofiber scaffolds fabricated from polyamide 6/12 (PA6/12). The nanofibers were prepared by the original needleless electrospun Nanospider technology, which enables to create nanofibers of defined diameter, porosity, and a basis weight. Copolymer PA6/12 was selected on the basis of the stability of its nanofibers in aqueous solutions, its biocompatibility, and its superior properties as a matrix for the growth of LSCs, MSCs, and corneal epithelial and endothelial cell lines. The morphology, growth properties, and viability of cells grown on PA6/12 nanofibers were comparable with those grown on plastic. LSCs labeled with the fluorescent dye PKH26 and grown on PA6/12 nanofibers were transferred onto the damaged ocular surface, where their seeding and survival were monitored. Cotransfer of LSCs with MSCs, which have immunosuppressive properties, significantly inhibited local inflammatory reactions and supported the healing process. The results thus show that nanofibers prepared from copolymer PA6/12 represent a convenient scaffold for growth of LSCs and MSCs and transfer to treat SC deficiencies and various ocular surface injuries.

Immunoregulatory properties of mouse limbal stem cells.

Stem cells have been demonstrated in nearly all adult mammalian tissues and play a vital role in their physiological renewal and healing after injury. Due to their irreplaceable role in tissue repair, these cells had to develop mechanisms protecting them from deleterious inflammatory immune reactions and ensuring their increased resistance to various apoptosis-inducing agents. In this study, we demonstrate that a population of mouse limbal cells highly enriched for cells expressing markers and characteristics of limbal stem cells (LSCs) suppresses in a dose-dependent manner the proliferation of lymphocytes elicited by mitogens or TCR-triggering and significantly inhibits the production of proinflammatory cytokines by activated T cells. The suppression was mediated by soluble factor(s) and did not affect early cell activation. LSCs were even more suppressive than mesenchymal stem cells or natural regulatory T cells. In addition, the cells expressing markers and characteristics of LSC had significantly higher levels of mRNA for Fas ligand and for the antiapoptotic molecules Mcl-1, XIAP, and survivin than other limbal cell populations. LSCs were also more resistant to staurosporin-induced apoptotic cell death and to cell-mediated cytotoxic reaction than other limbal cells. Collectively, these results suggest that SC isolated from fresh adult limbal tissue possess immunomodulatory properties and inhibit proinflammatory immune reactions. Simultaneously, these cells express high levels of mRNA for antiapoptotic molecules, which can protect them against cell-mediated cytotoxic reactions and various apoptosis-inducing agents.

DNA vaccination with all-trans retinoic acid treatment induces long-term survival and elicits specific immune responses requiring CD4+ and CD8+ T-cell activation in an acute promyelocytic leukemia mouse model.

DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4(+) or CD8(+) cells abolished this effect. CD4(+) depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4(+) and CD8(+) subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4(+) cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester-based assays showed major histocompatibility complex-restricted APL-specific T cell-mediated immune responses. Sorted APL-specific CD8(+)CD107a(+) T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA-treated mice were shown to secrete interferon-gamma when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARalpha vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-gamma-producing and cytotoxic T cells in the leukemic mice.

Distinct regulatory roles of transforming growth factor-beta and interleukin-4 in the development and maintenance of natural and induced CD4+ CD25+ Foxp3+ regulatory T cells.

The development and function of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) are strictly regulated by cytokines. Here we show that transforming growth factor-beta (TGF-beta) and interleukin-4 (IL-4) play a crucial and antagonistic role in the development of Tregs. Additionally, these cytokines also have distinct effects on the maintenance of natural (nTregs) and antigen-induced (iTregs) Tregs. Using double-staining and tracking of proliferation of purified and carboxyflourescein succinimidyl ester (CFSE)-labelled mouse T-cell subpopulations we demonstrated that CD4(+) CD25(+) Foxp3(+) iTregs develop upon alloantigenic stimulation in the presence of TGF-beta exclusively from CD4(+) CD25(-) Foxp3(-) precursors. Both the induction of Foxp3 expression and Treg proliferation were prevented when the cells were stimulated in the presence of IL-4. By contrast, nTregs did not proliferate in the presence of the antigen and TGF-beta, and partially lost their Foxp3 expression. IL-4 not only prevented the development of iTregs, but also down-regulated the level of Foxp3 mRNA and decreased the number of Foxp3(+) cells in a population of iTregs. Further analyses proved that IL-4 decreased the expression of Foxp3 only in a population of iTregs, whereas it substantially supported the survival of nTregs. Functional experiments showed that Tregs induced in the presence of alloantigen and TGF-beta inhibited, on a per-cell basis, cell proliferation comparably to nTregs, and their suppressive capacity was not modulated by IL-4. These data suggest that TGF-beta and IL-4 differentially regulate the development of Tregs and distinctly sustain Foxp3 expression and the number of nTregs and iTregs, but have no influence on the suppressive activity of Tregs on a per-cell basis.

A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient.

PURPOSE: To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse. METHODS: Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro. RESULTS: Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer. CONCLUSIONS: These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12(-)p63(+) population being closer to the primitive limbal SC.

Frequent antibody production against RARalpha in both APL mice and patients.

In an acute promyelocytic leukemia (APL)-transplantable mouse model, we previously reported the presence of antibodies recognizing PML-RARalpha and RARalpha in the sera of ATRA-treated mice. To evaluate this immune response, we determined the prevalence of anti-RARalpha antibodies in a cohort of 48 APL mice, treated by ATRA (n = 24) or by placebo pellets (n = 24), and in a preliminary subset of 9 patients with APL using a specific enzyme-linked immunosorbent assay (ELISA). In APL mice, significantly higher antibody levels were observed at the latest time points (day 48 to 58 levels superior to day 15 to 18 or day 28 to 38 levels). Antibody levels were higher in ATRA-treated mice than in placebo-treated mice and were also predictive of better survival. In the patients with APL, anti-RARalpha antibodies were detected at diagnosis and after maintenance therapy, reminiscent of the ATRA-treated APL mice. Antinuclear or antineutrophil cytoplasmic autoantibodies were also detected. These data reveal for the first time that in patients with APL an immune response may be detected at diagnosis and enhanced after maintenance therapy.