Cell Mitogenic Responses Research Articles

Aging alters the cell cycle control and mitogenic signaling responses of human hematopoietic stem cells

AbstractHuman hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38−CD45RA−CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor–stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.

Pneumonitis in patients with thymoma and Good's syndrome.

e20595 Background: The association between thymoma (T) and immunological dysregulations is well acknowledged. Good’s syndrome (GS), which occurs in approximately 6% to 11% of T patients, is characterized by hypogammaglobulinemia, few or absent B-cells (CD19+), CD4 T cells lymphopenia, abnormal CD4/CD8 T ratio and impaired T cell mitogenic response. Patients with GS have increased susceptibility to bacterial, opportunistic and viral infections related to both humoral and cell-mediated immunodeficiency. The recent Sars-Cov-2 pandemic drew attention to the clinical condition of fatal viral pneumonitis and cytokines storm. Here we reported our monocentric experience of pneumonitis in patients with T and GS before the Sars-Cov-2 pandemic. Methods: We conducted a retrospective analysis of T patients with associated GS referred to the Rare Tumours Reference Center of University Federico II of Naples over a 10-year period (from 2009 to 2019). All the patients with radiological and/or clinical pneumonitis diagnosis were evaluated for this report. Immunological features, histopathological diagnosis and clinical outcome were registered. Results: A total of 41 patients with T and GS were identified, including 17 patients with local disease (stage I-II according to Masaoka-Koga) and 24 with advanced disease (stage III-IV). The majority (56.3%) had B2 T diagnosis. Radiological and/or clinical pneumonitis diagnosis was assessed in 23 cases (56%). Viral pneumonitis was detected in 8 patients: 3 patients with H1N1 infection, 3 patients with CMV infection, 2 patients with EBV infection. Bacterial pneumonitis was diagnosed in 9 patients (3 patients with K. pneumoniae, 4 patients with S. aureus, 2 patients with H. influenza). Opportunistic pneumonitis was found in 2 patients with Aspergillosis infection. In 2 cases no pathogenic agent was identified. The immunophenotyping, assessed in 4 patients with viral pneumonitis, displayed very low/undetectable levels of B cells, with median % value of CD3+T cells and NKT of respectively 9.8% and 0.4% of the total leukocytes. The median % of Treg was 4.7%. CD4+/CD8+ ratio was variable, ranging from 1,2 to 0,6. Interestingly the number of B cells was extremely low, independent of CD4+/CD8+ ratio. Blood levels of cytokines, chemokines and growth factors revealed elevated IL-4, Eotaxin, CCL2 / MCP-1 and CCL5 / RANTES ad strong reduction of IL-10. PDGF-BB levels were also elevated. 15 patients required admission to intensive care Six patients died for fatal pneumonitis. Conclusions: The management of T patients with GS is extremely challenging. Clear diagnostic algorithms do not yet exist and immune-profiling and quantitative immunoglobulins should be considered a part of diagnostic search in these patients. The coexistence of cancer, infections and immunosuppression may trigger life-threatening conditions, such as fatal pneumonitis, which often require intensive care and multidisciplinary approach.

Treatment with Modified Extracts of the Microalga Planktochlorella nurekis Attenuates the Development of Stress-Induced Senescence in Human Skin Cells.

More recently, we have proposed a safe non-vector approach to modifying the biochemical profiles of the microalga Planktochlorella nurekis and obtained twelve clones with improved content of lipids and selected pigments and B vitamins and antioxidant activity compared to unaffected cells. In the present study, the biological activity of water and ethanolic extracts of modified clones is investigated in the context of their applications in the cosmetic industry and regenerative medicine. Extract-mediated effects on cell cycle progression, proliferation, migration, mitogenic response, apoptosis induction, and oxidative and nitrosative stress promotion were analyzed in normal human fibroblasts and keratinocytes in vitro. Microalgal extracts did not promote cell proliferation and were relatively non-cytotoxic when short-term treatment was considered. Long-term stimulation with selected microalgal extracts attenuated the development of oxidative stress-induced senescence in skin cells that, at least in part, was correlated with nitric oxide signaling and increased niacin and biotin levels compared to an unmodified microalgal clone. We postulate that selected microalgal extracts of Planktochlorella nurekis can be considered to be used in skin anti-aging therapy.

Mitochondrial Electron Transport Chain-Derived Superoxide Exits Macrophages: Implications for Mononuclear Cell-Mediated Pathophysiological Processes.

The involvement of mitochondrial electron transport chain (METC)-derived superoxide anion radical in cell protooncogene activation, mitogenic responses, and cancerous growth has recently received much attention. In order for METC-derived superoxide to participate in any of the above processes, its exit from mitochondria would be a critical step. Detection of intracellular superoxide showed that mitochondrial respiration is the major source of cellular superoxide in unstimulated or resting monocytes/macrophages. However, direct evidence for the exit of superoxide from mitochondria is presently lacking. Here we show that METC-derived superoxide does exit from mitochondria in unstimulated monocytes/macrophages. Release of superoxide was first found to occur with substrate-supported mitochondria isolated from these cells. We also observed the presence of extracellular superoxide with the intact unstimulated/resting cells. Extracellular superoxide was markedly diminished (>90%) by the mitochondrial inhibitor, rotenone, or the uncoupler, carbonylcyanide p-(trifluromethy) phenylhydrazone. Furthermore, cells with a deficient METC exhibited significant reduction (>90%) in extracellular superoxide, demonstrating that with intact cells METC-derived superoxide not only exits from mitochondria, but can be released extracellularly. Superoxide anion radical released from mitochondria could react with exogenous nitric oxide, forming peroxynitrite. Mitochondria-derived extracellular superoxide could also oxidize low-density lipoprotein (LDL). These results thus resolve any uncertainty on the ability of superoxide to exit from mitochondria. This study for the first time also identifies mitochondria as the major source of extracellular superoxide in unstimulated resting monocytes/macrophages, which has implications for the involvement of these mononuclear cells in various pathophysiological situations.

The class-specific BCR tonic signal modulates lymphomagenesis in a c-myc deregulation transgenic model.

Deregulation of c-myc by translocation onto immunoglobulin (Ig) loci can promote B cell malignant proliferations with phenotypes as diverse as acute lymphoid leukemia, Burkitt lymphoma, diffuse large B cell lymphoma, myeloma... The B cell receptor (BCR) normally providing tonic signals for cell survival and mitogenic responses to antigens, can also contribute to lymphomagenesis upon sustained ligand binding or activating mutations. BCR signaling varies among cell compartments and BCR classes. For unknown reasons, some malignancies associate with expression of either IgM or class-switched Ig. We explored whether an IgA BCR, with strong tonic signaling, would affect lymphomagenesis in c-myc IgH 3'RR transgenic mice prone to lymphoproliferations. Breeding c-myc transgenics in a background where IgM expression was replaced with IgA delayed lymphomagenesis. By comparison to single c-myc transgenics, lymphomas from double mutant animals were more differentiated and less aggressive, with an altered transcriptional program. Larger tumor cells more often expressed CD43 and CD138, which culminated in a plasma cell phenotype in 10% of cases. BCR class-specific signals thus appear to modulate lymphomagenesis and may partly explain the observed association of specific Ig classes with human B cell malignancies of differential phenotype, progression and prognosis.

The double bromodomain protein Brd2 promotes B cell expansion and mitogenesis

Bromodomain-containing transcriptional regulators represent new epigenetic targets in different hematologic malignancies. However, bromodomain-mediated mechanisms that couple histone acetylation to transcription in lymphopoiesis and govern mature lymphocyte mitogenesis are poorly understood. Brd2, a transcriptional coregulator that contains dual bromodomains and an extraterminal domain (the BET family), couples chromatin to cell-cycle progression. We reported previously the first functional characterization of a BET protein as an effector of mammalian mitogenic signal transduction: Eμ-Brd2 Tg mice develop "activated B cell" diffuse large B cell lymphoma. No other animal models exist for genetic or lentiviral expression of BET proteins, hampering testing of novel anti-BET anticancer drugs, such as JQ1. We transduced HSCs with Brd2 lentivirus and reconstituted recipient mice to test the hypothesis that Brd2 regulates hematopoiesis in BM and mitogenesis in the periphery. Forced expression of Brd2 provides an expansion advantage to the donor-derived B cell compartment in BM and increases mature B cell mitogenic responsiveness in vitro. Brd2 binds the cyclin A promoter in B cells, shown by ChIP, and increases cyclin A mRNA and protein levels, and S-phase progression in vitro in mitogen-stimulated primary B cells, but not T cells, reinforcing results from Eμ-Brd2 mice. The small molecule BET inhibitor JQ1 reduces B cell mitogenesis, consistent with the interpretation that BET inhibitors are antiproliferative. Brd2-specific knockdown experiments show that Brd2 is also required for hematopoiesis. We conclude that Brd2 plays a critical, independent role in regulation of mitogenic response genes, particularly cyclin A, in B cells.

Optimizing the cryopreservation of murine splenocytes for improved antigen-specific T cell function in ELISPOT

ELISPOT assays are routinely used to measure immune responses of T cells in fresh and frozen splenocytes preparations; however, standardized methods for murine ELISPOT are not widely available. Freezing cells can significantly impact the function of T cells. The aim of this study was to optimize cryopreservation protocols to retain antigen-specific T cell function at similar levels as freshly isolated T cells using murine splenocytes. We examined four factors that might have an impact on cell viability and function: freezing medium, resting cells prior to freezing, temperature of the medium for initial dilution of cells after thawing and resting of cells prior to ELISPOT. FVB/N mice were vaccinated with adjuvant only (CFA/IFA) or IGF-IR peptides, previously shown to be immunogenic by ELISPOT analysis. Mouse splenocytes were cryopreserved using five different media: Medium 1 (50% X-Vivo media, 40% FBS, 10% DMSO), Medium 2 (25% RMPI, 65% FBS, 10% DMSO), Medium 3 (90% FBS, 10% DMSO), Medium 4 (Amresco Media) and Medium 5 (EZ-CP2 Media). Prior to freezing, the cells were either rested on ice for 5 hours or immediately frozen and moved to liquid nitrogen. After four weeks, we used media at 37°C or 4°C for initial dilution of cells after thawing. The thawed cells were either rested overnight at 37°C or not rested. The recovery efficiency and T cell function were evaluated by determining cell viability, background levels, peptide responses, and mitogen responses in ELISPOT assays, respectively. We observed significantly lower cell viability when using freezing Medium 4 and 5, compared to the other media tested (Medium 5 vs Media 1-4: P<0.001; Medium 4 vs 1: P<0.001, Medium 4 vs 2 P<0.05). Resting cells for 5 hours prior to freezing resulted in higher viability, however this difference was not significant (P=0.072). Using media warmed to 37°C to dilute thawed samples resulted in significantly higher cell viability as compared to using media at 4°C (P<0.0001). Media 2 and 3 gave significantly lower background than Fresh cells (P<0.05). Medium 3 was the only freezing medium to result in similar levels of IGF1R peptide responses compared to Fresh. Overnight resting of cryopreserved cells before ELISPOT gave significantly lower T cells responses in both PHA and PMA/ionomycin controls compared to unrested cryopreserved cells (P<0.001).However, overnight resting of cells gave mitogen responses most similar to that of fresh cells when PHA or PMA/ionomycin controls were used. The method of cryopreservation can have a tremendous impact upon splenocytes viability and function. By using an optimized cryopreservation protocol, it is possible to obtain antigen-specific T cell function at levels similar to freshly isolated cells.

Abstract 4730: The double bromodomain protein Brd2 controls B cell expansion and mitogenesis.

Abstract Background: Bromodomain-containing coregulators of transcription have been identified recently as new epigenetic targets to treat a variety of hematologic malignancies, including acute myeloid leukemias. However, the bromodomain mechanisms that couple histone acetylation to transcription in lymphopoiesis, and that govern mature lymphocyte responsiveness to growth signals, are only partly understood. Brd2 is a transcriptional co-regulator that contains dual Bromodomains and an ExtraTerminal domain (defining the BET family) that couples chromatin modification to cell cycle progression. We previously reported the first functional characterization of a BET protein as an effector of mammalian mitogenic signal transduction: Eμ-Brd2 transgenic mice develop an ‘activated B cell’ type of diffuse large B cell lymphoma. There have been no other animal models reported for genetic or lentiviral expression of BET proteins, which hampers testing of novel anticancer drugs such as JQ1 and its derivatives. Methods: Here we use Brd2 lentivirus transduction of hematopoietic stem cells and reconstitution of lethally irradiated recipient mice to examine the function of Brd2 in hematopoiesis in the bone marrow and mitogenesis in the periphery. Results: Three-fold over-expression of Brd2 in hematopoietic stem cells selectively leads to a doubling of the B cell compartment and increases mature B cell mitogenic responsiveness ex vivo. Knockdown of Brd2 by shRNA totally ablates hematopoietic lineages, likely by inhibiting the lineage-committed progenitor expansion through down-regulation of cyclin A, which reinforces results from Eμ-Brd2 transgenic mice. The novel small molecule BET inhibitor JQ1 reduces B cell mitogenesis by about 60% in parallel experiments, but this effect likely extends to other related BET family members and lacks Brd2 specificity. Conclusion/Significance: The Brd2 pathway integrates lymphopoiesis, leukemogenesis and normal mitogenesis. Targeting this pathway with therapeutic drugs may therefore confer multiple, diverse benefits in the treatment of hematologic malignancy, B cell mediated disease and likely inflammation. Citation Format: Anna C. Belkina, Wanda P. Blanton, Frank L. Lombardi, Gerald V. Denis. The double bromodomain protein Brd2 controls B cell expansion and mitogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4730. doi:10.1158/1538-7445.AM2013-4730

Assessment of the Effect of Nilotinib (Tasigna) Maintenance Therapy After Allogeneic Stem Cell Transplantation in Patients with Advanced CML and Ph+ ALL On Immune Reconstitution and Lymphocyte Function

Abstract Abstract 4478 Allogeneic stem cell transplantation (AlloSCT) is the treatment of choice in advanced chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). However, post transplant relapse rate is high and outcome is often poor in this setting. Reduction of tumor mass pre-transplant and maintenance therapy post alloSCT, may improve response rate and reduce relapse rate. We speculated that the second-generation tyrosine-kinase inhibitor (TKI) Nilotinib (Tasigna, Novartis Pharmaceuticals) would be effective in achieving these goals. In the current study Nilotinib, was administered as maintenance therapy post alloSCT in patients (pts) with advanced CML and Ph+ ALL (study CAMN107AIL03T). However, TKIs have been demonstrated in previously published literature to affect T cells proliferation and signal transduction and to potentiate LGL and NK cell activity. Furthermore, in recent studies TKIs have also been demonstrated to ameliorate chronic GVHD. We therefore assessed immunological reconstitution and function including flow analysis of lymphocyte subsets, T-mitogenic response to αCD3 and PHA, thymic activity as determined by the quantification of the T cell receptor excision circles (TREC), TCR repertoire and NK cells cytotoxic activity against K562 cell line. In all, the study included 24 pts. Patients engrafted in a median day +15 (range, 10–38) with 100% donor chimerism. Acute GVHD grade 3/4 was reported in 3 pts (14%) and the rate of extensive chronic GVHD at last follow up was 50%. At 6 months after alloSCT, 11 of 15 pts with advanced CML had attained CCyR, 11 of 15 pts with advanced CML had attained a MMR or better, and 5 of 7 pts with Ph+ ALL attained a CR. The median OS was 16 months, with predicted 1- and 2- year rates of 55% (95% CI, 32% – 72%) and 50% (95% CI, 28% – 68%), respectively. The median PFS was 11 months, with predicted 1- and 2- year rates of 50% (95% CI, 28% – 68%) and 38% (95% CI, 17% – 59%), respectively. Immunological testing was performed pre- and post Nilotinib maintenance therapy in 12 pts (advanced CML-8, Ph+ ALL-4) who received Nilotinib for at least 90 days following alloSCT. The median age was 34.5 years (range, 21–57) and 75% were males. Six pts underwent alloSCT from an HLA-matched sibling donor, 4 from matched unrelated and 2 from an alternative donor (cord blood-1, haploidentical-1). All had myeloablative conditioning. GVHD prophylaxis included CSA and MMF. The relative percentage of T- lymphocyte subsets (assessed by FACS) and total lymphocytes number were stable during Nilotinib maintenance administration after alloSCT, while a 7.8±2.5 fold increase in B cells was noted. T cell mitogenic response with αCD3 and PHA (stimulation index ratio) was sustained (2.5±1.0, vs. 2.8±1.05 and 3.3±1.3 vs. 5.3±2.9 stimulation, pre- and post Nilotinib therapy, respectively). Mean thymic output determined by TREC quantification pre-, during and post Nilotinib administration was 81.8±108, 81.2±90.3 and 142.8±197.4 copies per 0.5ug DNA indicating continuous thymopoiesis. Similarly, no significant change of the TCR repertoire was observed during Nilotinib treatment. Specifically, normal expression of the TCR repertoire was detected in 15.1±5.5 and 15.3±5.6 of the examined TCRs, clonal expression was detected in 2.5±2.2 and 2.9±3 of the examined receptors, while reduced expression was detected only in 6.4±4.3 and 5.8±4.5 of the examined receptors pre-and post Nilotinib treatment, respectively. NK cytotoxic activity against K562 expressed as fold of change from baseline, also remained stable during Nilotinib treatment (2.8±1.1 and 2.3±0.8, respectively). In summary, Nilotinib maintenance therapy post alloSCT in pts with advanced CML and Ph+ALL did not interfere or jeopardized immune reconstitution and function including the number of immune cell subsets, T cell mitogenic response, TCR repertoire, thymic output and NK cytolytic activity post alloSCT. Based on this immunological data we would further recommend Nilotinib maintenance therapy post alloSCT in pts with advanced CML and Ph+ALL. Disclosures: Nagler: Novartis: Honoraria, Research Funding.

Assessment of the Effect of Nilotinib (Tasigna) Maintenance Therapy After Allogeneic Stem Cell Transplantation in Patients with Advanced CML and Ph+ ALL On Immune Reconstitution and Lymphocyte Function

AbstractAbstract 4478Allogeneic stem cell transplantation (AlloSCT) is the treatment of choice in advanced chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). However, post transplant relapse rate is high and outcome is often poor in this setting. Reduction of tumor mass pre-transplant and maintenance therapy post alloSCT, may improve response rate and reduce relapse rate. We speculated that the second-generation tyrosine-kinase inhibitor (TKI) Nilotinib (Tasigna, Novartis Pharmaceuticals) would be effective in achieving these goals. In the current study Nilotinib, was administered as maintenance therapy post alloSCT in patients (pts) with advanced CML and Ph+ ALL (study CAMN107AIL03T). However, TKIs have been demonstrated in previously published literature to affect T cells proliferation and signal transduction and to potentiate LGL and NK cell activity. Furthermore, in recent studies TKIs have also been demonstrated to ameliorate chronic GVHD. We therefore assessed immunological reconstitution and function including flow analysis of lymphocyte subsets, T-mitogenic response to αCD3 and PHA, thymic activity as determined by the quantification of the T cell receptor excision circles (TREC), TCR repertoire and NK cells cytotoxic activity against K562 cell line. In all, the study included 24 pts. Patients engrafted in a median day +15 (range, 10–38) with 100% donor chimerism. Acute GVHD grade 3/4 was reported in 3 pts (14%) and the rate of extensive chronic GVHD at last follow up was 50%. At 6 months after alloSCT, 11 of 15 pts with advanced CML had attained CCyR, 11 of 15 pts with advanced CML had attained a MMR or better, and 5 of 7 pts with Ph+ ALL attained a CR. The median OS was 16 months, with predicted 1- and 2- year rates of 55% (95% CI, 32% – 72%) and 50% (95% CI, 28% – 68%), respectively. The median PFS was 11 months, with predicted 1- and 2- year rates of 50% (95% CI, 28% – 68%) and 38% (95% CI, 17% – 59%), respectively. Immunological testing was performed pre- and post Nilotinib maintenance therapy in 12 pts (advanced CML-8, Ph+ ALL-4) who received Nilotinib for at least 90 days following alloSCT. The median age was 34.5 years (range, 21–57) and 75% were males. Six pts underwent alloSCT from an HLA-matched sibling donor, 4 from matched unrelated and 2 from an alternative donor (cord blood-1, haploidentical-1). All had myeloablative conditioning. GVHD prophylaxis included CSA and MMF. The relative percentage of T- lymphocyte subsets (assessed by FACS) and total lymphocytes number were stable during Nilotinib maintenance administration after alloSCT, while a 7.8±2.5 fold increase in B cells was noted. T cell mitogenic response with αCD3 and PHA (stimulation index ratio) was sustained (2.5±1.0, vs. 2.8±1.05 and 3.3±1.3 vs. 5.3±2.9 stimulation, pre- and post Nilotinib therapy, respectively). Mean thymic output determined by TREC quantification pre-, during and post Nilotinib administration was 81.8±108, 81.2±90.3 and 142.8±197.4 copies per 0.5ug DNA indicating continuous thymopoiesis. Similarly, no significant change of the TCR repertoire was observed during Nilotinib treatment. Specifically, normal expression of the TCR repertoire was detected in 15.1±5.5 and 15.3±5.6 of the examined TCRs, clonal expression was detected in 2.5±2.2 and 2.9±3 of the examined receptors, while reduced expression was detected only in 6.4±4.3 and 5.8±4.5 of the examined receptors pre-and post Nilotinib treatment, respectively. NK cytotoxic activity against K562 expressed as fold of change from baseline, also remained stable during Nilotinib treatment (2.8±1.1 and 2.3±0.8, respectively). In summary, Nilotinib maintenance therapy post alloSCT in pts with advanced CML and Ph+ALL did not interfere or jeopardized immune reconstitution and function including the number of immune cell subsets, T cell mitogenic response, TCR repertoire, thymic output and NK cytolytic activity post alloSCT. Based on this immunological data we would further recommend Nilotinib maintenance therapy post alloSCT in pts with advanced CML and Ph+ALL. Disclosures:Nagler:Novartis: Honoraria, Research Funding.

A Defect of the INK4-Cdk4 Checkpoint and Myc Collaborate in Blastoid Mantle Cell Lymphoma–Like Lymphoma Formation in Mice

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a monoclonal proliferation of lymphocytes with the co-expression of CD5 and CD43, but not of CD23. Typical MCL is associated with overexpression of cyclin D1, and blastoid MCL variants are associated with Myc (alias c-myc) translocations. In this study, we developed a murine model of MCL-like lymphoma by crossing Cdk4(R24C) mice with Myc-3'RR transgenic mice. The Cdk4(R24C) mouse is a knockin strain that expresses a Cdk4 protein that is resistant to inhibition by p16(INK4a) as well as other INK4 family members. Ablation of INK4 control on Cdk4 does not affect lymphomagenesis, B-cell maturation, and functions in Cdk4(R24C) mice. Additionally, B cells were normal in numbers, cell cycle activity, mitogen responsiveness, and Ig synthesis in response to activation. By contrast, breeding Cdk4(R24C) mice with Myc-3'RR transgenic mice prone to develop aggressive Burkitt lymphoma-like lymphoma (CD19(+)IgM(+)IgD(+) cells) leads to the development of clonal blastoid MCL-like lymphoma (CD19(+)IgM(+)CD5(+)CD43(+)CD23(-) cells) in Myc/Cdk4(R24C) mice. Western blot analysis revealed high amounts of Cdk4/cyclin D1 complexes as the main hallmark of these lymphomas. These results indicate that although silent in nonmalignant B cells, a defect in the INK4-Cdk4 checkpoint can participate in lymphomagenesis in conjunction with additional alterations of cell cycle control, a situation that might be reminiscent of the development of human blastoid MCL.

Impact of Fresh or Used Litter on the Posthatch Immune System of Commercial Broilers

This study was carried out to investigate the effects of exposure of growing broiler chickens of commercial origin to used poultry litter on intestinal and systemic immune responses. The litter types evaluated were fresh wood shavings or used litter obtained from commercial poultry farms with or without a history of gangrenous dermatitis (GD). Immune parameters measured were serum nitric oxide (NO) levels, serum antibody titers against Eimeria or Clostridium perfringens, mitogen-induced spleen cell proliferation, and intestinal intraepithelial lymphocyte or splenic lymphocyte subpopulations. At 43 days posthatch, birds raised on used litter from a GD farm had higher serum NO levels and greater Eimeria or C. perfringens antibody levels compared with chickens raised on fresh litter or used, non-GD litter. Birds raised on non-GD and GD used litter had greater spleen cell mitogenic responses compared with chickens raised on fresh litter. Finally, spleen and intestinal lymphocyte subpopulations were increased or decreased depending on the litter type and the surface marker analyzed. Although it is likely that the presence of Eimeria oocysts and endemic viruses varies qualitatively and quantitatively between flocks and, by extension, varies between different used litter types, we believe that these data provide evidence that exposure of growing chicks to used poultry litter stimulates humoral and cell-mediated immune responses, presumably due to contact with contaminating enteric pathogens.

Maintenance Therapy with Nilotinib (Tasigna) Post Allogeneic Stem Cell Transplantation (AlloSCT) for Advanced (>CP1) Chronic Myeloid Leukemia (CML) and Ph+ Acute Lymphoblastic Leukemia (ALL)

Abstract 2011AlloSCT is the treatment of choice in advanced CML (>CP1) and in Ph+ ALL. However, the post transplant relapse rate is high and outcome is rather poor in this setting. Nilotinib (Tasigna, Novartis Pharmaceuticals) is a very effective, second generation, novel TKI. Reduction of tumor mass pre- and maintenance therapy post alloSCT, may improve response rate and reduce transplant related toxicities (TRT) and relapses. The aim of the current study (CAMN107AIL03T) was to investigate whether nilotinib administration pre-alloSCT (400mg BID) and at escalating doses of 200–400mg BID post-alloSCT will intensify remission, reduce relapse and improve outcome without increasing TRT in pts with advanced CML and Ph+ ALL. 24 pts (M-13, F-11) participated in the study. The median age was 36 years (range, 18–58). 17 pts were with advanced CML (BC-10, AP-7) and 7 with Ph+ ALL. Cytogenetic and molecular responses were assessed by FISH and RQ-PCR (Taqman, LeukemiaNet and Eutos criteria). 5 pts harbored ABL kinas domain mutations by Sequenom assay (Y253H-2, A397P-1, E255K-1, M351T-1). 9 pts received nilotinib (400mg BID) pre-alloSCT and 6 responded (2 are too early to evaluate) (BC- CHR–2, BC - CCR-2, AP - PCR-1, AP - CHR-1). 22/24 pts underwent alloSCT (2 are pending) from an HLA-matched sibling (n=11), matched unrelated (n=8) or alternative donor (CB-2, Haplo-1). All, but one, had myeloablative conditioning (Bu/Cy or Flu/Bu-14, TBI/Cy-7). Graft versus host disease (GVHD) prophylaxis included CSA and methotrexate or MMF. 21/22 pts engrafted in median day+13 (range, 9–38) with 100% donor chimerism. TRT included mucositis in all pts, encephalitis-2, pericarditis-1, VOD-1 and infection-3. 3 pts died very early post transplant from sepsis and multi organ failure. Acute GVHD ≥ Gr II was observed in 10 pts (III-IV in 5). Chronic GVHD was observed in 15 pts (extensive – 9). Sixteen of the 22 transplanted pts received nilotinib (200mg × 2/d – 10, 300mg × 2/d – 6) starting at median day +38 (range, 30–158) post alloSCT. 6 pts did not receive nilotinib post alloSCT due to early death -3, progressive disease -1, severe pancytopenia -1, refusal -1. No pt received the planned 400mg BID dose. In 9 pts nilotinib administration was delayed due to either liver abnormalities -3, severe thrombocytopenia -3 or infection -3. Nilotinib dose had to be reduced (n=4) or stopped (n=3) due to Gr III-IV non-hematological toxicities -5 pts, mainly abnormal liver function tests -4 in combination with pruritus -1, vomiting -1 and abdominal pain -1, or hematological toxicities -2 pts. In 1 pt we observed increased levels of amylase without clinical signs of pancreatitis. QTc (470 msec) prolongation was observed in 1 pt. All, but 2 pts achieved MMR post alloSCT and 6 of them (CML) converted to CMR following nilotinib therapy. Response was durable and maintained. Only in 1 pt with disease progression kinas mutation (G205E and F359V) were detected. With a median follow up of 14.5 months (range, 0.5–36) 12 pts are alive while 12 died (Infection -5, GVHD -3, TTP-1, disease progression -3). In 2 pts disease progressed but responded to further therapy. 5 of 6 pts that did not receive nilotinib died (early death post AllSCT -3, disease progression -1, GVHD Gr IV -1). Immunological evaluation pre- and post nilotinib administration disclosed no significant change in T, B and NK cell numbers and T cell mitogenic response to PHA and anti CD3. Thymic output (TREC) and receptor repertoire (Spectrotyping) analysis indicates continuous thymopoiesis in 8/13 evaluated pts. NK cytotoxic activity against K562 increased in 9/15 evaluated pts. In conclusion, post alloSCT nilotinib maintenance therapy in extremely high risk pts with advanced CML and Ph+ ALL may prevent relapse and disease progression as 1) pts achieved MMR and 6 converted to CMR with nilotinib therapy and 2) only 5/16 pts that received nilotinib post alloSCT progressed [in 4 of them nilotinib could not be administrated as per protocol because of side effects (n=2) or low compliance (n=2)]. Although a formal maximal tolerated dose (MTD) of nilotinib post alloSCT has not been reached, it seems that administration of nilotinib early post alloSCT may be associated with increased toxicity. nilotinib administration post alloSCT does not impair immunological reconstitution, while GVHD incidence seems not to be reduced. Based on these data we would recommend nilotinib maintenance therapy post alloSCT in pts with advanced CML and Ph+ ALL. Disclosures:Nagler:Novartis: Honoraria, Research Funding. Off Label Use: Nilotinib (Tasigna) for reduction of tumor mass pre- and maintenance therapy post alloSCT in patients with CML and Ph+ ALL (novel TKI).

Diazepam leads to enhanced severity of orthopoxvirus infection and immune suppression

Benzodiazepines are drugs widely used as tranquilizers and in various other indications. We treated Balb/c mice with diazepam and infected them with cowpox (CPXV) and vaccinia virus (VACV). Disease index, weight loss and the antibody response were determined. Additionally the influence of different benzodiazepines on the mitogen response of human peripheral blood lymphocytes and spleen cells was tested. Diazepam led to earlier disease onset, prolonged duration of symptoms, higher weight loss and overall disease index in VACV infected mice. CPXV infected mice developed poxviral skin lesions only after drug administration and a significant decrease in the specific antibody response was also observed. Diazepam and alprazolam also inhibited the proliferative response of human lymphocytes/spleen cells in vitro but did not show noteworthy apoptotic effects. It is surprising that even a single dose of diazepam has a profound influence on the immune system, sufficient to facilitate symptomatic infectious disease. These data provide first evidence that commonly used drugs like Valium ® may augment severity of rare poxvirus infections such as CPXV or monkeypox. As VACV is still used as life vaccine against smallpox there is also a risk of enhanced side effects or possible interference with the success of vaccination.

Deletion of the Met receptor in the collecting duct decreases renal repair following ureteral obstruction

Hepatocyte growth factor and its receptor, Met, activate biological pathways necessary for repair and regeneration following kidney injury. The Met receptor is expressed in multiple cell types within the kidney, each of which is capable of regulating fibrotic responses. To specifically address the role of the Met receptor in the adult collecting duct during renal injury, a conditional knockout mouse (Met(fl/fl);HoxB7-Cre) was generated and tested using unilateral ureteral obstruction, a model of nephron injury, fibrosis, and repair. Following obstruction in these mice there was increased expression of collagens I and IV along with plasminogen activator inhibitor 1, a known regulator of matrix degradation, compared to ureteral obstructed non-flox littermates. There were trends toward increased interstitial fibrosis, infiltration of the interstitium, and acute tubular necrosis in the knockout mice despite similar degrees of hydronephrosis to the control littermates. The Met(fl/fl);HoxB7-Cre mice; however, had reduced tubular cell proliferation and kidney regenerative capacity after release of the obstruction, thus leading to diminished functional recovery. We suggest that Met receptor signaling in the collecting duct acts as a major regulator of cell survival and propagation of the repair process with a possible secondary role to diminish inflammatory and fibrotic responses.

CpG-Induced Myeloid CD11b+Gr-1+ Cells Efficiently Suppress T Cell–Mediated Immunoreactivity and Graft-Versus-Host Disease in a Murine Model of Allogeneic Cell Therapy

Transplantation of mismatched allografts in irradiated recipients results in lethal graft- versus-host disease (GVHD). In our study, pretransplantation donor treatment with CpG, administered either alone or emulsified in incomplete Freund's adjuvant, efficiently prevented GVHD in sublethally irradiated recipients of haploidentical (H-2b into H-2b/d) and fully mismatched (H-2b into H-2d) allografts. CpG treatment of donor mice caused an accumulation of double-positive CD11bGr-1 cells in their blood and spleens, whereas treatment with CpG+IFA resulted in an even greater accumulation of these cells. Isolated CD11b+ cells from the spleens of CpG+IFA-treated mice efficiently suppressed alloreactivity in vitro (> 92%), as determined by co-culturing these cells in mixed lymphocyte reactions. After CpG+IFA treatment, a T cell–depleted fraction enriched with CD11b+Gr-1+ cells, acting as myeloid suppressor cells, was able to efficiently prevent GVHD induced by naïve T cells in the sublethally irradiated recipients: 20/21 mice remained GVHD-free survivors for more than 200 days. Splenocytes from CpG+IFA-treated mice displayed enhanced interleukin (IL)-6, IL-10, and interferon-γ production, reduced T cell allogeneic and mitogenic responses, as well as failure of T cells to induce GVHD. In summary, CpG treatment led to impaired T cell function, enriched myeloid suppressor cells and regulatory cytokine production, which together appear to suppress alloreactivity and protect against the development of GVHD. We hypothesize that similar immunoregulatory effects could be applied experimentally in a clinical setting when inhibition of alloreactivity is required in recipients of stem cell allografts.

Impaired cellular immune response in harbour seals (Phoca vitulina) feeding on environmentally contaminated herring.

In a 2.5-year immunotoxicological study, two groups of captive harbour seals (Phoca vitulina) were fed herring from the heavily polluted Baltic Sea or from the relatively uncontaminated Atlantic Ocean. Blood samples were collected at regular intervals, and functional immunological parameters were monitored. T cell mitogen and mixed lymphocyte-induced proliferative responses of peripheral blood mononuclear cells (PBMC) obtained from seals fed Baltic herring were significantly reduced over the course of experiment. Upon immunization with rabies virus antigen (RV) and tetanus toxoid (TT), specific proliferative responses of PBMC from the seals fed Baltic herring were also significantly reduced. Impairment of T cell-mediated immune responses became especially apparent during the second year on the respective diets, and correlated significantly to 2,3,7,8-tetrachloro-dibenzo-p-dioxin toxic equivalent levels in blubber biopsies taken from the seals after 2 years on the respective diets. Humoral immune responses, including lipopolysaccharide (LPS)-induced lymphoproliferative responses, in vitro immunoglobulin production by PBMC, as well as RV-, TT-and poliovirus-specific serum antibody responses following immunization, remained largely unaffected. We conclude that suppression of the cellular immune response in the seals fed Baltic herring was induced by the chronic exposure to immunotoxic environmental contaminants accumulated through the food chain. Since cellular immune responses are known to be of crucial importance in the clearance of morbillivirus infections, these results suggest that environmental pollution-related immunosuppression may have contributed to the severity and extent of recent morbillivirus-related mass mortalities among marine mammals.

Immunorestoration and augmentation of mitogen lymphocyte response in Graffi tumor bearing hamsters by purified saponin mixture from Astragalus corniculatus

Investigations on the effect of purified saponin mixture (PSM) obtained from the aerial parts of Astragalus corniculatus Bieb. (Fabaceae) on mitogen response of the spleen cells in Graffi tumor bearing (GTBH) and healthy hamsters were reported. The saponin mixture in a doses of 50mg/kg b.w. was injected i.p. 4 times starting simultaneously with implantation of tumor cells. Stimulation indices to phytohemagglutinine (PHA) and lipopolysaccharide (LPS) of lymphocytes in spleens of tumor bearing hamsters (TBH) were significantly decreased during the whole period of the observation. It was established that PSM stimulated the functions of spleen cells in Graffi-TBH, resulting in increased mitogen response to PHA and LPS. The stimulation was better expressed in healthy PSM-treated hamsters. The proliferation response of spleen lymphocytes to PSM was also found. PSM did not change the in vitro proliferation ability of Graffi tumor cells. The results obtained proved the immunostimulating and immunorestorating activity of PSM on the T- and B-spleen cells in healthy and GTBH hamsters, as well as the proliferative response of it to PSM.

The Effects of Fumonisin B1 on viability and mitogenic response of avian immune cells

Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides (formerly Fusarium moniliforme) and is found in diverse crops such as corn, wheat, and barley. Many diseases linked to FB1, such as porcine pulmonary edema, rat hepatic cancer, and equine leukoencephalomalacia, indicate a compromised immune system. The purpose of this study was to determine whether FB1 altered immunological responses in various cell populations of Single Comb White Leghorn chicks. Cells collected for this study were obtained from those immunological organs with well-defined responses (i.e., spleen, thymus, and blood). Cell populations were exposed to 5 to 50 µg/mL FB1 in vitro for 24 to 72 h, and viability and mitogenic response were evaluated. The effects of FB1 on the mitogenic response were evaluated in cell populations from the spleen and blood stimulated with the mitogens, lipopolysaccharide, concanavalin A, and pokeweed mitogen and in thymocytes stimulated with concanavalin A. The 3-(4,5-dimethylthazol-2-yl)-diphenyl-2H-tetrazolium bromide (MTT) reduction assay was used to assess viability and mitogenic response. Fumonisin B1 decreased spleen cell viability and mitogenic response, albeit the degree of decrease varied with mitogen and time of exposure. Fumonisin B1 increased number of viable thymic cells at 50 µg/mL but had no effect on the mitogenic response of thymocytes. Fumonisin B1 had no effect on blood lymphocyte viability or mitogenic response.

The immune response in autism: a new frontier for autism research

Autism spectrum disorders (ASD) are part of a broad spectrum of neurodevelopmental disorders known as pervasive developmental disorders, which occur in childhood. They are characterized by impairments in social interaction, verbal and nonverbal communication and the presence of restricted and repetitive stereotyped behaviors. At the present time, the etiology of ASD is largely unknown, but genetic, environmental, immunological, and neurological factors are thought to play a role in the development of ASD. Recently, increasing research has focused on the connections between the immune system and the nervous system, including its possible role in the development of ASD. These neuroimmune interactions begin early during embryogenesis and persist throughout an individual's lifetime, with successful neurodevelopment contingent upon a normal balanced immune response. Immune aberrations consistent with a dysregulated immune response, which so far, have been reported in autistic children, include abnormal or skewed T helper cell type 1 (T(H)1)/T(H)2 cytokine profiles, decreased lymphocyte numbers, decreased T cell mitogen response, and the imbalance of serum immunoglobulin levels. In addition, autism has been linked with autoimmunity and an association with immune-based genes including human leukocyte antigen (HLA)-DRB1 and complement C4 alleles described. There is potential that such aberrant immune activity during vulnerable and critical periods of neurodevelopment could participate in the generation of neurological dysfunction characteristic of ASD. This review will examine the status of the research linking the immune response with ASD.