Integrin/TGF-ß1 Inhibitor GLPG-0187 Blocks SARS-CoV-2 Delta and Omicron Pseudovirus Infection of Airway Epithelial Cells In Vitro, Which Could Attenuate Disease Severity.

As COVID-19 continues to pose major risk for vulnerable populations, including the elderly, immunocompromised, patients with cancer, and those with contraindications to vaccination, novel treatment strategies are urgently needed. SARS-CoV-2 infects target cells via RGD-binding integrins, either independently or as a co-receptor with surface receptor angiotensin-converting enzyme 2 (ACE2). We used pan-integrin inhibitor GLPG-0187 to demonstrate the blockade of SARS-CoV-2 pseudovirus infection of target cells. Omicron pseudovirus infected normal human small airway epithelial (HSAE) cells significantly less than D614G or Delta variant pseudovirus, and GLPG-0187 effectively blocked SARS-CoV-2 pseudovirus infection in a dose-dependent manner across multiple viral variants. GLPG-0187 inhibited Omicron and Delta pseudovirus infection of HSAE cells more significantly than other variants. Pre-treatment of HSAE cells with MEK inhibitor (MEKi) VS-6766 enhanced the inhibition of pseudovirus infection by GLPG-0187. Because integrins activate transforming growth factor beta (TGF-ß) signaling, we compared the plasma levels of active and total TGF-ß in COVID-19+ patients. The plasma TGF-ß1 levels correlated with age, race, and number of medications upon presentation with COVID-19, but not with sex. Total plasma TGF-ß1 levels correlated with activated TGF-ß1 levels. Moreover, the inhibition of integrin signaling prevents SARS-CoV-2 Delta and Omicron pseudovirus infectivity, and it may mitigate COVID-19 severity through decreased TGF-ß1 activation. This therapeutic strategy may be further explored through clinical testing in vulnerable and unvaccinated populations.

Integrin/TGF-ß1 inhibitor GLPG-0187 blocks SARS-CoV-2 Delta and Omicron pseudovirus infection of airway epithelial cells which could attenuate disease severity.

As COVID-19 continues to pose major risk for vulnerable populations including the elderly, immunocompromised, patients with cancer, and those with contraindications to vaccination, novel treatment strategies are urgently needed. SARS-CoV-2 infects target cells via RGD-binding integrins either independently or as a co-receptor with surface receptor angiotensin-converting enzyme 2 (ACE2). We used pan-integrin inhibitor GLPG-0187 to demonstrate blockade of SARS-CoV-2 pseudovirus infection of target cells. Omicron pseudovirus infected normal human small airway epithelial (HSAE) cells significantly less than D614G or Delta variant pseudovirus, and GLPG-0187 effectively blocked SARS-CoV-2 pseudovirus infection in a dose-dependent manner across multiple viral variants. GLPG-0187 inhibited Omicron and Delta pseudovirus infection of HSAE cells more significantly than other variants. Pre-treatment of HSAE cells with MEK inhibitor (MEKi) VS-6766 enhanced inhibition of pseudovirus infection by GLPG-0187. Because integrins activate TGF-ß signaling, we compared plasma levels of active and total TGF-ß in COVID-19+ patients. Plasma TGF-ß1 levels correlated with age, race, and number of medications upon presentation with COVID-19, but not with sex. Total plasma TGF-ß1 levels correlated with activated TGF-ß1 levels. In our preclinical studies, Omicron infects lower airway lung cells less efficiently than other COVID-19 variants. Moreover, inhibition of integrin signaling prevents SARS-CoV-2 Delta and Omicron pseudovirus infectivity, and may mitigate COVID-19 severity through decreased TGF-ß1 activation. This therapeutic strategy may be further explored through clinical testing in vulnerable and unvaccinated populations.

Effects of Interleukin-1ß in Glycinergic Transmission at the Central Amygdala.

Interleukin-1ß (IL-1ß) is an important cytokine that modulates peripheral and central pain sensitization at the spinal level. Among its effects, it increases spinal cord excitability by reducing inhibitory Glycinergic and GABAergic neurotransmission. In the brain, IL-1ß is released by glial cells in regions associated with pain processing during neuropathic pain. It also has important roles in neuroinflammation and in regulating NMDA receptor activity required for learning and memory. The modulation of glycine-mediated inhibitory activity via IL-1ß may play a critical role in the perception of different levels of pain. The central nucleus of the amygdala (CeA) participates in receiving and processing pain information. Interestingly, this nucleus is enriched in the regulatory auxiliary glycine receptor (GlyR) ß subunit (ßGlyR); however, no studies have evaluated the effect of IL-1ß on glycinergic neurotransmission in the brain. Hence, we hypothesized that IL-1ß may modulate GlyR-mediated inhibitory activity via interactions with the ßGlyR subunit. Our results show that the application of IL-1ß (10 ng/ml) to CeA brain slices has a biphasic effect; transiently increases and then reduces sIPSC amplitude of CeA glycinergic currents. Additionally, we performed molecular docking, site-directed mutagenesis, and whole-cell voltage-clamp electrophysiological experiments in HEK cells transfected with GlyRs containing different GlyR subunits. These data indicate that IL-1ß modulates GlyR activity by establishing hydrogen bonds with at least one key amino acid residue located in the back of the loop C at the ECD domain of the ßGlyR subunit. The present results suggest that IL-1ß in the CeA controls glycinergic neurotransmission, possibly via interactions with the ßGlyR subunit. This effect could be relevant for understanding how IL-1ß released by glia modulates central processing of pain, learning and memory, and is involved in neuroinflammation.

RGD-binding integrins and TGF-ß in SARS-CoV-2 infections - novel targets to treat COVID-19 patients?

The new coronavirus SARS-CoV-2 is a global pandemic and a severe public health crisis. SARS-CoV-2 is highly contagious and shows high mortality rates, especially in elderly and patients with pre-existing medical conditions. At the current stage, no effective drugs are available to treat these patients. In this review, we analyse the rationale of targeting RGD-binding integrins to potentially inhibit viral cell infection and to block TGF-ß activation, which is involved in the severity of several human pathologies, including the complications of severe COVID-19 cases. Furthermore, we demonstrate the correlation between ACE2 and TGF-ß expression and the possible consequences for severe COVID-19 infections. Finally, we list approved drugs or drugs in clinical trials for other diseases that also target the RGD-binding integrins or TGF-ß. These drugs have already shown a good safety profile and, therefore, can be faster brought into a trial to treat COVID-19 patients.

Adipose tissue macrophages as a therapeutic target in obesity-associated diseases.

Obesity is an increasing problem in developed and developing countries. Individuals with obesity have a higher risk of several diseases, such as cardiovascular disease, increased risk of insulin resistance, type 2 diabetes, infertility, degenerative disorders, and also certain types of cancer. Adipose tissue (AT) is considered an extremely active endocrine organ, and the expansion of AT is accompanied by the infiltration of different types of immune cells, which induces a state of low-grade, chronic inflammation and metabolic dysregulation. Even though the exact mechanism of this low-grade inflammation is not fully understood, there is clear evidence that AT-infiltrating macrophages (ATMs) play a significant role in the pro-inflammatory state and dysregulated metabolism. ATMs represent the most abundant class of leukocytes in AT, constituting 5% of the cells in AT in individuals with normal weight. However, this percentage dramatically increases up to 50% in individuals with obesity, suggesting an important role of ATMs in obesity and its associated complications. In this review, we discuss current knowledge of the function of ATMs during steady-state and obesity and analyze its contribution to different obesity-associated diseases, highlighting the potential therapeutic target of ATMs in these pathological conditions.

The Emerging Therapeutic Potential of Nitro Fatty Acids and Other Michael Acceptor-Containing Drugs for the Treatment of Inflammation and Cancer.

Nitro fatty acids (NFAs) are endogenously generated lipid mediators deriving from reactions of unsaturated electrophilic fatty acids with reactive nitrogen species. Furthermore, Mediterranean diets can be a source of NFA. These highly electrophilic fatty acids can undergo Michael addition reaction with cysteine residues, leading to post-translational modifications (PTM) of selected regulatory proteins. Such modifications are capable of changing target protein function during cell signaling or in biosynthetic pathways. NFA target proteins include the peroxisome proliferator-activated receptor γ (PPAR-γ), the pro-inflammatory and tumorigenic nuclear factor-κB (NF-κB) signaling pathway, the pro-inflammatory 5-lipoxygenases (5-LO) biosynthesis pathway as well as soluble epoxide hydrolase (sEH), which is essentially involved in the regulation of vascular tone. In several animal models of inflammation and cancer, the therapeutic efficacy of well-tolerated NFA has been demonstrated. This has already led to clinical phase II studies investigating possible therapeutic effects of NFA in subjects with pulmonary arterial hypertension. Albeit Michael acceptors feature a broad spectrum of bioactivity, they have for a rather long time been avoided as drug candidates owing to their presumed unselective reactivity and toxicity. However, targeted covalent modification of regulatory proteins by Michael acceptors became recognized as a promising approach to drug discovery with the recent FDA approvals of the cancer therapeutics, afatanib (2013), ibrutinib (2013), and osimertinib (2015). Furthermore, the Michael acceptor, neratinib, a dual inhibitor of the human epidermal growth factor receptor 2 and epidermal growth factor receptor, was recently approved by the FDA (2017) and by the EMA (2018) for the treatment of breast cancer. Finally, a number of further Michael acceptor drug candidates are currently under clinical investigation for pharmacotherapy of inflammation and cancer. In this review, we focus on the pharmacology of NFA and other Michael acceptor drugs, summarizing their potential as an emerging class of future antiphlogistics and adjuvant in tumor therapeutics.

Ion Channel Function During Oocyte Maturation and Fertilization.

The proper maturation of both male and female gametes is essential for supporting fertilization and the early embryonic divisions. In the ovary, immature fully-grown oocytes that are arrested in prophase I of meiosis I are not able to support fertilization. Acquiring fertilization competence requires resumption of meiosis which encompasses the remodeling of multiple signaling pathways and the reorganization of cellular organelles. Collectively, this differentiation endows the egg with the ability to activate at fertilization and to promote the egg-to-embryo transition. Oocyte maturation is associated with changes in the electrical properties of the plasma membrane and alterations in the function and distribution of ion channels. Therefore, variations on the pattern of expression, distribution, and function of ion channels and transporters during oocyte maturation are fundamental to reproductive success. Ion channels and transporters are important in regulating membrane potential, but also in the case of calcium (Ca2+), they play a critical role in modulating intracellular signaling pathways. In the context of fertilization, Ca2+ has been shown to be the universal activator of development at fertilization, playing a central role in early events associated with egg activation and the egg-to-embryo transition. These early events include the block of polyspermy, the completion of meiosis and the transition to the embryonic mitotic divisions. In this review, we discuss the role of ion channels during oocyte maturation, fertilization and early embryonic development. We will describe how ion channel studies in Xenopus oocytes, an extensively studied model of oocyte maturation, translate into a greater understanding of the role of ion channels in mammalian oocyte physiology.

Drug-Mediated Intracellular Donation of Nitric Oxide Potently Inhibits 5-Lipoxygenase: A Possible Key to Future Antileukotriene Therapy.

AIMS: 5-Lipoxygenase (5-LO) is the key enzyme of leukotriene (LT) biosynthesis and is critically involved in a number of inflammatory diseases such as arthritis, gout, bronchial asthma, atherosclerosis, and cancer. Because 5-LO contains critical nucleophilic amino acids, which are sensitive to electrophilic modifications, we determined the consequences of a drug-mediated intracellular release of nitric oxide (NO) on 5-LO product formation by human granulocytes and on 5-LO-dependent pulmonary inflammation in vivo. RESULTS: Clinically relevant concentrations of NO-releasing nonsteroidal anti-inflammatory drugs and other agents releasing NO intracellularly suppress 5-LO product synthesis in isolated human granulocytes via direct S-nitrosylation of 5-LO at the catalytically important cysteines 416 and 418. Furthermore, suppression of 5-LO product formation was observed in ionophore-stimulated human whole blood and in an animal model of pulmonary inflammation. INNOVATION: Here, we report for the first time that drugs releasing NO intracellularly are efficient 5-LO inhibitors in vitro and in vivo at least equivalent to approved 5-LO inhibitors. CONCLUSION: Our findings provide a novel mechanistic strategy for the development of a new class of drugs suppressing LT biosynthesis by site-directed nitrosylation. The results may also help to better understand the well-recognized anti-inflammatory clinically relevant actions of NO-releasing drugs. Furthermore, our study describes in detail a novel molecular mode of action of NO. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Angel Lanas, Hartmut Kühn, Joan Clària, Orina Belton. Antioxid. Redox Signal. 28, 1265-1285.

Combined Anti-VEGF and Anti-CTLA-4 Therapy Elicits Humoral Immunity to Galectin-1 Which Is Associated with Favorable Clinical Outcomes.

The combination of anti-VEGF blockade (bevacizumab) with immune checkpoint anti-CTLA-4 blockade (ipilimumab) in a phase I study showed tumor endothelial activation and immune cell infiltration that were associated with favorable clinical outcomes in patients with metastatic melanoma. To identify potential immune targets responsible for these observations, posttreatment plasma from long-term responding patients were used to screen human protein arrays. We reported that ipilimumab plus bevacizumab therapy elicited humoral immune responses to galectin-1 (Gal-1), which exhibits protumor, proangiogenesis, and immunosuppressive activities in 37.2% of treated patients. Gal-1 antibodies purified from posttreatment plasma suppressed the binding of Gal-1 to CD45, a T-cell surface receptor that transduces apoptotic signals upon binding to extracellular Gal-1. Antibody responses to Gal-1 were found more frequently in the group of patients with therapeutic responses and correlated with improved overall survival. In contrast, another subgroup of treated patients had increased circulating Gal-1 protein instead, and they had reduced overall survival. Our findings suggest that humoral immunity to Gal-1 may contribute to the efficacy of anti-VEGF and anti-CTLA-4 combination therapy. Gal-1 may offer an additional therapeutic target linking anti-angiogenesis and immune checkpoint blockade. Cancer Immunol Res; 5(6); 446-54. ©2017 AACR.

Soluble PD-L1 as a Biomarker in Malignant Melanoma Treated with Checkpoint Blockade.

Blockade of the pathway including programmed death-ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) has produced clinical benefits in patients with a variety of cancers. Elevated levels of soluble PD-L1 (sPD-L1) have been associated with worse prognosis in renal cell carcinoma and multiple myeloma. However, the regulatory roles and function of sPD-L1 particularly in connection with immune checkpoint blockade treatment are not fully understood. We identified four splice variants of PD-L1 in melanoma cells, and all of them are secreted. Secretion of sPD-L1 resulted from alternate splicing activities, cytokine induction, cell stress, cell injury, and cell death in melanoma cells. Pretreatment levels of sPD-L1 were elevated in stage IV melanoma patient sera compared with healthy donors. High pretreatment levels of sPD-L1 were associated with increased likelihood of progressive disease in patients treated by CTLA-4 or PD-1 blockade. Although changes in circulating sPD-L1 early after treatment could not distinguish responders from those with progressive disease, after five months of treatment by CTLA-4 or PD-1 blockade patients who had increased circulating sPD-L1 had greater likelihood of developing a partial response. Induction of sPD-L1 was associated with increased circulating cytokines after CTLA-4 blockade but not following PD-1 blockade. Circulating sPD-L1 is a prognostic biomarker that may predict outcomes for subgroups of patients receiving checkpoint inhibitors. Cancer Immunol Res; 5(6); 480-92. ©2017 AACR.

Vaccination with autologous myeloblasts admixed with GM-K562 cells in patients with advanced MDS or AML after allogeneic HSCT.

We report a clinical trial testing vaccination of autologous myeloblasts admixed with granulocyte-macrophage colony-stimulating factor secreting K562 cells after allogeneic hematopoietic stem cell transplantation (HSCT). Patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with ≥5% marrow blasts underwent myeloblast collection before HSCT. At approximately day +30, 6 vaccines composed of irradiated autologous myeloblasts mixed with GM-K562 were administered. Tacrolimus-based graft-versus-host disease (GVHD) prophylaxis was not tapered until vaccine completion (∼day 100). Thirty-three patients with AML (25) and MDS (8) enrolled, 16 (48%) had ≥5% marrow blasts at transplantation. The most common vaccine toxicity was injection site reactions. One patient developed severe eosinophilia and died of eosinophilic myocarditis. With a median follow-up of 67 months, cumulative incidence of grade 2-4 acute and chronic GVHD were 24% and 33%, respectively. Relapse and nonrelapse mortality were 48% and 9%, respectively. Progression-free survival (PFS) and overall survival (OS) at 5 years were 39% and 39%. Vaccinated patients who were transplanted with active disease (≥5% marrow blasts) had similar OS and PFS at 5 years compared with vaccinated patients transplanted with <5% marrow blasts (OS, 44% vs 35%, respectively, P = .81; PFS, 44% vs 35%, respectively, P = .34). Postvaccination antibody responses to angiopoietin-2 was associated with superior OS (hazard ratio [HR], 0.43; P = .031) and PFS (HR, 0.5; P = .036). Patients transplanted with active disease had more frequent angiopoeitin-2 antibody responses (62.5% vs 20%, P = .029) than those transplanted in remission. GM-K562/leukemia cell vaccination induces biologic activity, even in patients transplanted with active MDS/AML. This study is registered at www.clinicaltrials.gov as #NCT 00809250.

Michael acceptor containing drugs are a novel class of 5-lipoxygenase inhibitor targeting the surface cysteines C416 and C418.

Recently, we published that nitro-fatty acids (NFA) are potent electrophilic molecules which inhibit 5-lipoxygenase (5-LO) by interacting catalytically with cysteine residues next to a substrate entry channel. The electrophilicity is derived from an intramolecular Michael acceptor moiety consisting of an electron-withdrawing group in close proximity to a double bond. The potential of the Michael acceptor moiety to interact with functionally relevant cysteines of proteins potentially renders them effective and sustained enzyme activity modulators. We screened a large library of naturally derived and synthetic electrophilic compounds to investigate whether other types of Michael acceptor containing drugs suppress 5-LO enzyme activity. The activity was measured by assessing the effect on the 5-LO product formation of intact human polymorphonuclear leukocytes. We demonstrated that a number of structurally different compounds were suppressive in the activity assays and showed that Michael acceptors of the quinone and nitro-alkene group produced the strongest inhibition of 5-LO product formation. Reactivity with the catalytically relevant cysteines 416 and 418 was confirmed using mutated recombinant 5-LO and mass spectrometric analysis (MALDI-MS). In the present study, we show for the first time that a number of well-recognized naturally occurring or synthetic anti-inflammatory compounds carrying a Michael acceptor, such as thymoquinone (TQ), the paracetamol metabolite NAPQI, the 5-LO inhibitor AA-861, and bardoxolone methyl (also known as RTA 402 or CDDO-methyl ester) are direct covalent 5-LO enzyme inhibitors that target the catalytically relevant cysteines 416 and 418.

Vaccination with Irradiated Autologous Tumor Cells Mixed with Irradiated GM-K562 Cells Stimulates Antitumor Immunity and T Lymphocyte Activation in Patients with Recurrent Malignant Glioma.

PURPOSE: Recurrent malignant glioma carries a dismal prognosis, and novel therapies are needed. We examined the feasibility and safety of vaccination with irradiated autologous glioma cells mixed with irradiated GM-K562 cells in patients undergoing craniotomy for recurrent malignant glioma. EXPERIMENTAL DESIGN: We initiated a phase I study examining the safety of 2 doses of GM-K562 cells mixed with autologous cells. Primary endpoints were feasibility and safety. Feasibility was defined as the ability for 60% of enrolled subjects to initiate vaccination. Dose-limiting toxicity was assessed via a 3+3 dose-escalation format, examining irradiated tumor cells mixed with 5 × 10(6) GM-K562 cells or 1 × 10(7) GM-K562 cells. Eligibility required a priori indication for resection of a recurrent high-grade glioma. We measured biological activity by measuring delayed type hypersensitivity (DTH) responses, humoral immunity against tumor-associated antigens, and T-lymphocyte activation. RESULTS: Eleven patients were enrolled. Sufficient numbers of autologous tumor cells were harvested in 10 patients, all of whom went on to receive vaccine. There were no dose-limiting toxicities. Vaccination strengthened DTH responses to irradiated autologous tumor cells in most patients, and vigorous humoral responses to tumor-associated angiogenic cytokines were seen as well. T-lymphocyte activation was seen with significantly increased expression of CTLA-4, PD-1, 4-1BB, and OX40 by CD4(+) cells and PD-1 and 4-1BB by CD8(+) cells. Activation was coupled with vaccine-associated increase in the frequency of regulatory CD4(+) T lymphocytes. CONCLUSIONS: Vaccination with irradiated autologous tumor cells mixed with GM-K562 cells is feasible, well tolerated, and active in patients with recurrent malignant glioma. Clin Cancer Res; 22(12); 2885-96. ©2016 AACR.

Regulation of tumorigenic Wnt signaling by cyclooxygenase-2, 5-lipoxygenase and their pharmacological inhibitors: A basis for novel drugs targeting cancer cells?

Canonical Wnt signaling is a highly conserved pathway with a prominent role in embryogenic development, adult tissue homeostasis, cell polarization, stem cell biology, cell differentiation, and proliferation. Furthermore, canonical Wnt signaling is of pivotal importance in the pathogenesis of a number of cancer types and crucially affects tumor initiation, cancer cell proliferation, cancer cell apoptosis, and metastasis. Reports over the last decade have provided strong evidence for a pathophysiological role of Wnt signaling in non-malignant classical inflammatory and neurodegenerative diseases. Although, several agents suppressing the Wnt pathway at different levels have been identified, the development of clinically relevant Wnt-inhibiting agents remains challenging due to selectivity and toxicity issues. Several studies have shown that long-term administration of non-steroidal anti-inflammatory drugs protects against colon cancer and potentially other tumor types by interfering both with the COX and the Wnt pathway. Our own studies have shown that non-steroidal anti-inflammatory drugs suppress Wnt signaling by targeting the pro-inflammatory enzyme 5-lipoxygenase which is the key enzyme pathophysiologically involved in the synthesis of leukotrienes. Furthermore, we found a direct link between the 5-lipoxygenase and Wnt signaling pathways, which is essential for the maintenance of leukemic stem cells. Accordingly, genetic and pharmacological inhibition of 5-lipoxygenase led to an impairment of Wnt-dependent acute and chronic myeloid leukemic stem cells. We believe that 5-lipoxygenase inhibitors might represent a novel type of Wnt inhibitor activating a potentially naturally occurring novel mechanism of suppression of Wnt signaling that is non-toxic, at least in mice, and is potentially well tolerated in patients.

Angiogenic cytokines are antibody targets during graft-versus-leukemia reactions.

PURPOSE: The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT). EXPERIMENTAL DESIGN: A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT. RESULTS: A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival. CONCLUSIONS: These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias.

Characterization of HLA-DR-restricted T-cell epitopes derived from human proteinase 3.

Human proteinase 3 (PRTN3) is a leukemia-associated antigen specifically recognized by CD8+ cytotoxic T-lymphocytes (CTL). PRTN3 also has been shown to elicit both antibody responses and T-cell proliferation in patients with Wegener's granulomatosis. In order to improve current vaccines that aim to stimulate CTL without inducing harmful autoimmune disease, it is necessary to study the role of PRTN3-specific CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Since both TH and Treg cells recognize antigens in the context of HLA-class-II-molecules, identification of HLA-class-II-associated peptide-epitopes from self-antigens such as PRTN3 is required. Here, we analyzed T-cell responses against proteinase 3 using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. We first screened a panel of ten epitope peptide candidates selected with the TEPITOPE program and found that nine out of ten peptides induced PRTN3 peptide-specific proliferation of T-cells with precursor frequencies of 0-1.1 x 10(-6). For one peptide-epitope, PRTN3(235), T-cell-clones were demonstrated to be capable of recognizing naturally processed protein antigen in a HLA-DR-restricted fashion. PRTN3(235)-specific T-cells could be stimulated from the blood of healthy individuals with multiple HLA-DR-genotypes. In summary, the identified PRTN3(235)-epitope can be used to study the role of CD4+ TH- and Treg-cells in immune responses against PRTN3 in leukemia patients and patients with Wegener's disease.

Identification of a promiscuous HLA DR-restricted T-cell epitope derived from the inhibitor of apoptosis protein survivin.

The inhibitor of apoptosis protein survivin is a promising tumor-associated antigen specifically recognized by CD8+ cytotoxic effector T-lymphocytes (CTL). To improve current vaccines that aim to induce survivin-specific CTL, it is necessary to study the role of CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Because both TH and Treg cells recognize antigens in the context of HLA-class II molecules, identification of HLA class II-associated peptide epitopes from survivin is required. Here, we analyzed T-cell responses against survivin using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. Six peptides were shown to induce CD4+ T-cell responses, restricted by HLA-DR molecules. For one peptide epitope, SVN10, T-cell clones were demonstrated to be capable of recognizing naturally processed antigen. SVN10-specific T cells could be stimulated from the blood of healthy individuals and cancer patients with multiple HLA-DR genotypes. Thus the identified SVN10 epitope can be used to study the role of CD4+ TH and Treg cells in immune responses and possibly be included in a multivalent peptide vaccine against survivin.

Casein kinase 1 delta (CK1delta) interacts with the SNARE associated protein snapin.

In this study we identified snapin as an interaction partner of the CK1 isoform delta (CK1delta) in the yeast two-hybrid system and localized the interacting domains of both proteins. The interaction of CK1delta with snapin was confirmed by co-immunoprecipitation. Snapin was phosphorylated by CK1delta in vitro. Both proteins localized in close proximity in the perinuclear region, wherein snapin was found to associate with membranes of the Golgi apparatus. The identification of snapin as a new substrate of CK1delta points towards a possible function for CK1delta in modulating snapin specific functions.

Innate immunity mediated by TLR9 modulates pathogenicity in an animal model of multiple sclerosis.

Inflammatory diseases of the CNS, such as MS and its animal model EAE, are characterized by infiltration of activated lymphocytes and phagocytes into the CNS. Within the CNS, activation of resident cells initiates an inflammatory cascade, leading to tissue destruction, demyelination, and neurologic deficit. TLRs recognize microbes and are pivotal mediators of innate immunity. Within the CNS, augmented TLR expression during EAE is observed, even in the absence of any apparent microbial involvement. To determine the functional relevance of this phenomenon during sterile autoimmunity, we studied the role of different TLRs as well as their common signaling adaptor MyD88 in the development of EAE. We found that MyD88 mice were completely EAE resistant. Surprisingly, this protection is partly due to engagement of the CpG receptor TLR9. Restricting the MyD88 or TLR9 mutation to host radio-resistant cells, including the cells within the CNS, revealed that engagement of radio-resistant cells modulated the disease course and histopathological changes. Our data clearly demonstrate that both TLR9 and MyD88 are essential modulators of the autoimmune process during the effector phase of disease and suggest that endogenous "danger signals" modulate the disease pathogenesis.

Gene transfer into human T lymphocytes and natural killer cells by Ad5/F35 chimeric adenoviral vectors.

OBJECTIVE: Genetic modification of effector lymphocytes, such as T cells and natural killer (NK) cells, is essential for many approaches to gene-based immunotherapy of cancer. However, transduction of lymphocytes has proven difficult by currently available gene transfer methods. Previous studies have shown that chimeric fiber-modified Ad5/F35 adenoviral vectors are able to efficiently transduce hematopoietic cells including immature progenitors. In this study, we examined the gene transfer into T lymphocytes and NK cells using Ad5/F35 compared with conventional Ad5 adenovectors. METHODS: Primary T and NK cells were isolated from healthy donors' peripheral blood leukocytes by immunomagnetic selection. Cell lines and primary lymphocytes were transduced with replication-defective Ad5/F35 and Ad5, both containing a GFP reporter gene under the control of a CMV promoter. Transduction efficiencies were monitored by flow cytometry. The function of transduced lymphocytes was assessed by analysis of proliferative responses to mitogenic agents and in mixed leukocyte reactions. RESULTS: Transgene expression was detected in up to 45% of primary CD3+ T lymphocytes and in up to 60% of primary NK cells using Ad5/F35. In contrast, conventional Ad5 transduced less than 8% and 5% of primary T cells and NK cells, respectively. Transduction efficiencies were similar in CD4+ and CD8+ T lymphocytes, and transgene expression could be detected for up to seven days. Activation of T cells significantly enhanced the efficiency of Ad5/F35-mediated gene transfer. Adenoviral transduction of lymphocytes did not result in any impairment of proliferative functions. CONCLUSION: The results of this study demonstrate that both T lymphocytes and NK cells can be transduced by chimeric Ad5/F35 adenoviral vectors.