Hernan Concha Quezada

Autologous antitumor activity by NK cells expanded from myeloma patients using GMP-compliant components

Multiple myeloma (MM) is an incurable plasma cell malignancy with poor outcome. The most promising therapeutic options currently available are combinations of transplantation, targeted pharmacotherapy, and immunotherapy. Cell-based immunotherapy after hematopoietic stem-cell transplantation has been attempted, but with limited efficacy. Natural killer (NK) cells are interesting candidates for new means of immunotherapy; however, their potential clinical use in MM has not been extensively studied. Here, we explored the possibility of expanding NK cells from the peripheral blood of 7 newly diagnosed, untreated MM patients, using good manufacturing practice (GMP)-compliant components. After 20 days of culture, the number of NK cells from these patients had expanded on average 1600-fold. Moreover, expanded NK cells showed significant cytotoxicity against primary autologous MM cells, yet retained their tolerance against nonmalignant cells. Based on these findings, we propose that autologous NK cells expanded ex vivo deserve further attention as a possible new treatment modality for MM.

Clinical-grade, large-scale, feeder-free expansion of highly active human natural killer cells for adoptive immunotherapy using an automated bioreactor

BACKGROUND AIMS Natural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion. METHODS Peripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality. RESULTS Significant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 x 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks. CONCLUSIONS These results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.

Attenuated EAN in TNF-α Deficient Mice Is Associated with an Altered Balance of M1/M2 Macrophages

The role of tumor necrosis factor (TNF)-α and its receptors in neuroautoimmune and neuroinflammatory diseases has been controversial. On the basis of our previous studies, we hereby aimed to further clarify TNF-α’s mechanism of action and to explore the potential role of TNF-α receptor (TNFR)1 as a therapeutic target in experimental autoimmune neuritis (EAN). EAN was induced by immunization with P0 peptide 180–199 in TNF-α knockout (KO) mice and anti-TNFR1 antibodies were used to treat EAN. Particularly, the effects of TNF-α deficiency and TNFR1 blockade on macrophage functions were investigated. The onset of EAN in TNF-α KO mice was markedly later than that in wild type (WT) mice. From day 14 post immunization, the clinical signs of TNF-α KO mice were significantly milder than those of their WT counterparts. Further, we showed that the clinical severity of WT mice treated with anti-TNFR1 antibodies was less severe than that of the control WT mice receiving PBS. Nevertheless, no difference with regard to the clinical signs of EAN or inflammatory infiltration in cauda equina was seen between TNF-α KO and WT mice with EAN after blockade of TNFR1. Although TNF-α deficiency did not alter the proliferation of lymphocytes in response to either antigenic or mitogenic stimuli, it down-regulated the production of interleukin (IL)-12 and nitric oxide (NO), and enhanced the production of IL-10 in macrophages. Increased ratio of regulatory T cells (Tregs) and reduced production of interferon (IFN)-γ in cauda equina infiltrating cells, and elevated levels of IgG2b antibodies against P0 peptide 180–199 in sera were found in TNF-α KO mice with EAN. In conclusion, TNF-α deficiency attenuates EAN via altering the M1/M2 balance of macrophages.

Circulating Th17, Th22, and Th1 Cells Are Elevated in the Guillain-Barré Syndrome and Downregulated by IVIg Treatments

The Guillain-Barré syndrome (GBS) is considered a T helper 1 (Th1) cells-mediated acute inflammatory peripheral neuropathy. However, some changes in GBS could not be explained completely by Th1 cells pathogenic role. Recently, Th17 cells have been identified and can mediate tissue inflammation and autoimmune response. Therefore, a study on the role of Th17 and Th22 cells and their cytokines in GBS is necessary for exploring the pathogenesis of GBS. Here, we detected the frequency of Th1, Th17, and Th22 cells by using 4-color flow cytometry and we detected the plasma levels of IL-17 and IL-22 by ELISA in GBS patients, relapsing-remitting multiple sclerosis patients at the acute phase of relapse, viral encephalitis or meningitis patients and healthy controls. Our data showed that the frequency of circulating Th1, Th17, and Th22 cells was significantly increased in GBS patients. The plasma levels of IL-17 and IL-22 in GBS and relapsing-remitting multiple sclerosis at the acute phase of relapse were also markedly elevated. Enhanced circulating Th22 cells were correlated with GBS severity. Intravenous immunoglobulin therapy downregulated Th17, and Th22 cells and the plasma levels of IL-17 and IL-22 in GBS patients. Th17 and Th22 cells may be involved in the pathogenesis of GBS, and intravenous immunoglobulin mediates therapeutic effects by downregulating these cells and their cytokines.

IL-18 deficiency aggravates kainic acid-induced hippocampal neurodegeneration in C57BL/6 mice due to an overcompensation by IL-12.

The role of interleukin-18 (IL-18) in excitotoxic neurodegeneration is largely unknown. To address this issue, we used kainic acid (KA)-induced hippocampal neurodegeneration in IL-18 knockout (KO) mice. One day after KA administration, clinical symptoms and histopathological changes did not differ between IL-18 KO mice and wild-type mice. However, 7 days after KA application the hippocampal neurodegeneration was markedly severe in IL-18 KO mice as demonstrated by increased locomotion and prominent histopathological changes including neuronal cell loss, microglia activation and astrogliosis. Surprisingly, when wild-type mice received recombinant mouse IL-18 (rmIL-18) in advance, after KA treatment both the clinical and pathological signs were dose-dependently aggravated compared to mice without rmIL-18 pre-treatment. To clarify the mechanism behind this, we assessed the expression of the IL-18 associated cytokines IL-12, IL-1beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) in the hippocampus by immunohistochemistry and flow cytometry. IL-12 and IFN-gamma expression was strongly increased in IL-18 KO mice when compared to wild-type mice 7 days after KA treatment in agreement with increased microglia activation. These results suggest that the role of IL-18 in excitotoxic injury in IL-18 deficient mice may be overcompensated by increased IL-12 secretion.

mTor is a signaling hub in cell survival: a mass-spectrometry-based proteomics investigation.

mTor plays a central role in controlling protein homeostasis and cell survival. Recently, we have demonstrated that perturbations of mTor signaling are implicated in Alzheimers disease (AD) and that mTor complex 1 (mTorC1) is involved in the formation of toxic phospho-tau. Therefore, we employed mass-spectrometry-based proteomics to identify specific protein expression changes in relation with cell survival in human neuroblastoma SH-SY5Y cells expressing genetically modified mTor. Cell death in SH-SY5Y cells was induced by moderate serum deprivation. Using flow cytometry we observed that up-regulated mTor complex 2 (mTorC2) increases the number of viable cells. By using a combination approach of proteomic and enrichment analysis we have identified several proteins (Thioredoxin-dependent peroxide reductase, Peroxiredoxin-5, Cofilin 1 (non-muscle), Annexin A5, Mortalin, and 14-3-3 protein zeta/delta) involved in mitochondrial integrity, apoptotosis, and pro-survival functions (caspase inhibitor activity and anti-apoptosis) that were significantly altered by mTor activity modulation. The major findings of this study are the implication of mTorC2 but not mTorC1 in cell viability modulation by activating the pro-survival machinery. Taken together, these results suggest that up-regulated mTorC2 might be playing an important role in promoting cell survival by suppressing the mitochondria-caspase-apoptotic pathway in vitro.

TNF‐α receptor 1 deficiency enhances kainic acid–induced hippocampal injury in mice

The exact role of TNF‐α in excitotoxic neurodegeneration of the brain is unclear. To address this issue, the kainic acid (KA)–induced hippocampal injury model, a well‐characterized model of human neurodegenerative diseases, was used in TNF‐α receptor 1 (TNFR1)–knockout (TNFR1−/−) mice in the present study. After nasal application of a single dose of 40 mg of KA per kilogram body weight, TNFR1−/− mice showed significantly more severe seizures than the wild‐type mice. In addition, obvious neurodegeneration, enhanced microglia activation, and astrogliosis in the hippocampus, as well as increased locomotor activity, were found in TNFR1−/− mice compared with the wild‐type controls 8 days after KA delivery. Moreover, CC chemokine receptor 3 expression on activated microglia was increased 3 days after KA treatment in TNFR1−/− mice, as measured by flow cytometry. These data suggest that TNF‐α may play a protective role through TNFR1 signaling.

Aggravation of experimental autoimmune neuritis in TNF-α receptor 1 deficient mice

Abstract The role of tumor necrosis factor (TNF)-α and its receptors in the pathogenesis of experimental autoimmune neuritis (EAN) induced by P0 peptide 180–199 in TNFR1 (p55) deficient (TNFR1−/−) mice was investigated. Compared to wild type EAN mice, TNFR1−/− EAN mice developed significantly more severe clinical signs, in parallel with enhanced numbers of inflammatory infiltrating cells in peripheral nerves and splenic P0-reactive T cell proliferation, as well as increased obviously MHC class II and CCR3 expression on the macrophages in the cauda equina. Our data indicated that TNF-α might have anti-inflammatory effect preventing the development of EAN in this mouse model.

TNF-α receptor 1 deficiency reduces antigen-presenting capacity of Schwann cells and ameliorates experimental autoimmune neuritis in mice

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic pro-inflammatory cytokine with potentially neurodestructive effects and plays a pivotal role in autoimmune demyelinating disease. To address the role of TNF-alpha in the pathogenesis of experimental autoimmune neuritis (EAN), the current study investigated the antigen-presenting capacity of Schwann cells (SCs) in EAN induced by P0 protein peptide 106-125 in TNF-alpha receptor 1 deficient (TNFR1(-/-)) mice. The antigen-presenting capacity of SCs was assessed by the expression of MHC class II (MHCII), CD40, CD80 and CD86 molecules on activated SCs as well as by induction of T cell proliferation in co-cultures of P0 protein peptide 106-125 specific T cells with activated SCs. In addition, the expression of inducible nitric oxide synthase (iNOS) was measured in activated SCs by flow cytometry. TNFR1(-/-) EAN mice developed significantly delayed and reduced clinical signs of EAN compared to wild type EAN mice. In parallel, the expression of MHCII, CD80 and iNOS on SCs were decreased in TNFR1(-/-) mice compared to wild type mice. Likewise, proliferation of P0 protein peptide 106-125 specific T cells simulated by activated SCs of TNFR1(-/-) EAN mice was lower than that of wild type EAN mice. Our data suggest that TNF-alpha may exert pro-inflammatory effects in EAN via TNFR1 by up-regulating the antigen-presenting function and iNOS production of SCs.

Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo

The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase‐1 (mPGES‐1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES‐1 inhibitors, we investigated the effect of aminothiazoles on inflammation‐induced PGE2 synthesis in vitro, using human gingival fibroblasts stimulated with the cytokine IL‐1β and a cell‐free mPGES‐1 activity assay, as well as on inflammation‐induced bone resorption in vivo, using ligature‐induced experimental periodontitis in Sprague‐Dawley rats. Aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) and 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644) reduced IL‐1β‐induced PGE2 production in fibroblasts (IC50 1.1 and 1.5 μM, respectively) as well as recombinant mPGES‐1 activity, without affecting activity or expression of the upstream enzyme cyclooxygenase‐2. In ligature‐induced experimental periodontitis, alveolar bone loss, assessed by X‐ray imaging, was reduced by 46% by local treatment with TH‐848, compared to vehicle, without any systemic effects on PGE2, 6‐keto PGF1α, LTB4 or cytokine levels. In summary, these results demonstrate that the aminothiazoles represent novel mPGES‐1 inhibitors for inhibition of PGE2 production and reduction of bone resorption in experimental periodontitis, and may be used as potential anti‐inflammatory drugs for treatment of chronic inflammatory diseases, including periodontitis.—Kats, A., Båge, T., Georgsson, P., Jönsson, J., Quezada, H. C., Gustafsson, A., Jansson, L., Lindberg, C., Näsström, K., Yucel‐Lindberg, T. Inhibition of microsomal prostaglandin E synthase‐1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo. FASEB J. 27, 2328–2341 (2013). www.fasebj.org