Pieter Spee - Academia.edu (original) (raw)

Papers by Pieter Spee

OncoImmunology, Apr 8, 2015

Annals of the Rheumatic Diseases, 2013

Conclusions: Our data indicate that mice deficient in mPGES-1 do not respond to vagus-mediated im... more Conclusions: Our data indicate that mice deficient in mPGES-1 do not respond to vagus-mediated immunosuppression. Moreover, we show immunosuppressant effects of PGE 2 on TNF release. These results implicate an involvement of PGE 2 as an important mediator in the cholinergic anti-inflammatory pathway, and provide support for further investigations regarding the interaction between cholinergic and prostaglandin systems.

Annals of the Rheumatic Diseases, 2014

ACR20, 50 and 70 response, remission (rem; defined as Clinical Disease Activity Index [CDAI] ≤2.8... more ACR20, 50 and 70 response, remission (rem; defined as Clinical Disease Activity Index [CDAI] ≤2.8, Simplified Disease Activity Index [SDAI] ≤3.3, Boolean score ≤1), low disease activity (LDA; CDAI ≤10, SDAI ≤11) or improvement in physical function (Health Assessment Questionnaire-Disability Index [HAQ-DI] >0.3) were assessed by disease duration and treatment subgroups. Results: 646 pts were randomized and treated with SC ABA (n=318) or ADA (n=328) on background MTX (71 and 70 pts with ≤6 months' disease duration; 247 and 258 pts with >6 months' disease duration). Baseline characteristics were balanced between the disease duration subgroups, with the exception of a higher percentage of males among pts with ≤6 months' disease duration treated with ADA compared with the other subgroups. Among pts with ≤6 months' disease duration, 23.9% (SC ABA) and 30.0% (ADA) of pts discontinued study treatment; 19.8% (SC ABA) and 24.0% (ADA) of pts with >6 months' disease duration discontinued. Clinical responses by disease duration at Years 1 (AMPLE primary endpoint) and 2 are summarized in the table. Conclusions: In contrast to what has been shown with DMARDs, data from this post hoc analysis of the AMPLE trial show that pts treated with effective biologic DMARDs (SC abatacept or adalimumab), whether treated early in the course of disease (≤6 months) or later, achieved comparable responses across a range of clinical measures.

Annals of the Rheumatic Diseases, 2013

ABSTRACT Background CD94/NKG2A+ CD56bright Natural Killer (NK) cells represent the majority of NK... more ABSTRACT Background CD94/NKG2A+ CD56bright Natural Killer (NK) cells represent the majority of NK cells in synovial fluid (SF) of Rheumatoid Arthritis (RA) patients and were proposed to exert an immunoregulatory role [1]. Prophylactic treatment with anti-NKG2A antibody fragment F(ab)’2 in murine collagen-induced arthritis reduced disease development by activating NK cells through blockade of its inhibitory receptor CD94/NKG2A [2]. Novo Nordisk A/S has generated an antagonistic, humanized anti-human NKG2A monoclonal antibody NNC141-0100 that blocks interaction between CD94/NKG2A and its ligand HLA-E. Objectives The aim of this study was to characterize the binding specificity of NNC141-0100 and the expression pattern of its target, the CD94/NKG2A receptors, in peripheral blood (PB), SF and/or synovial tissue from RA patients and healthy donors (HD). Methods CD94/NKG2A and HLA-E expression was investigated using flow cytometry, immunohistochemistry, digital image analysis and double-immunofluorescence (DIF) staining. Results In PB and SF from HD or RA patients, NNC141-0100 binding correlated with expression of CD94/NKG2A as detected by another anti-NKG2A mAb, and was restricted to subsets of NK and T cells. We observed a similar distribution of CD94/NKG2A on PB lymphocytes from HD and RA patients where ∼50% of NK cells expressed CD94/NKG2A. In contrast, the majority (>90%) of NK cells in RA SF were CD94/NKG2A+, confirming the previously reported disease-associated accumulation of CD94/NKG2A+ NK cells in RA SF [1]. CD94/NKG2A+ distribution on T cells was similar in PB from HD and RA patients and in RA SF (∼3%). In RA synovial tissue CD94/NKG2A was expressed by the majority of NK cells and a small subset of T cells. CD94/NKG2A+ cells were predominantly localized in lymphoid aggregates, but also present throughout RA synovium, whereas CD94/NKG2A+ cells were absent in synovium from HD. The CD94/NKG2A+ ligand, HLA-E, was detected on all infiltrating leukocytes in RA SF and synovium, at levels at least comparable to PB leucocytes from HD. Moreover, HLA-E was expressed by resident cells such as synoviocytes and endothelial cells in RA synovium. Conclusions These data demonstrate that CD94/NKG2A and its ligand HLA-E are expressed at sites of inflammation in RA. NNC141-0100 specifically binds to CD94/NKG2A+ NK cells accumulating in inflamed joints of RA patients, thus treatment with NNC141-0100 may promote the elimination of activated pro-inflammatory cells and suppress inflammation in RA patients. NNC141-0100 is currently being developed for the treatment of RA. Disclosure of Interest V. Pascal Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, Y. Sundström Grant/Research support from: Novo Nordisk A/S, A. Fasth Grant/Research support from: Novo Nordisk A/S, V. Malmström Grant/Research support from: Novo Nordisk A/S, L. Berg Grant/Research support from: Novo Nordisk A/S, P. Kvist Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, P. Spee Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, E. Galsgaard Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S

Annals of the Rheumatic Diseases, 2014

Methods: A549 cells were induced with IL-1b and treated with mPGES-1 inhibitor (compound III) or ... more Methods: A549 cells were induced with IL-1b and treated with mPGES-1 inhibitor (compound III) or COX-2 inhibitor (NS398). Eicosanoid profiles were analyzed using LC-MS/MS and protein profiles were analyzed using mass spectrometry based proteomics. FA composition of total lipids was determined using gas chromatography with flame ionization detector and sphingolipids were analysed by LC-MS/MS. Results: Prostanoid profiles in supernatants from the IL-1b-induced cells treated with the COX-2 or mPGES-1 inhibitors were significantly different suggesting diverse effects of the inhibitors on downstream pathways. Proteomic analysis of A549 cells identified the reduction of SCD levels in response to treatment with mPGES-1 or COX-2 inhibitors, as well as the suppression of the levels of a number of proteins involved in metabolism of fatty acids and sphingolipids. Conclusions: The results suggest that down-regulation of COX-2/mPGES-1/PGE2 axis affects the lipid metabolism in A549 cells. These effects have important implications regarding potential consequences of pharmacologic mPGES-1 inhibition.

Proceedings of the National Academy of Sciences of the United States of America, Aug 22, 2011

Acta dermato-venereologica, 2017

Interleukin-1α (IL-1α) and its receptor antagonist IL-1RA play a pivotal role in skin homeostasis... more Interleukin-1α (IL-1α) and its receptor antagonist IL-1RA play a pivotal role in skin homeostasis and disease. Although the use of biopsies to sample these cytokines from human skin is widely employed in dermatological practice, knowledge about less invasive, in vivo sampling methods is scarce. The aim of this study was to provide an overview of such methods by systematically reviewing studies in Medline, EMBASE, Web of Science and Cochrane Library using combinations of the terms "IL-1α", IL-1RA", "skin", "human", including all possible synonyms. Quality was assessed using the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) checklist. The search, performed on 14 October 2016, revealed 10 different sampling methods, with varying degrees of invasiveness and wide application spectrum, including assessment of both normal and diseased skin, from several body sites. The possibility to sample quantifiable amounts of cytokines from human skin with no or minimal discomfort holds promise for linking clinical outcomes to molecular profiles of skin inflammation.

Journal of Infectious Diseases and Therapy, Nov 2, 2016

Journal of Investigative Dermatology, Sep 1, 2016

Genetic deficiency or haploinsufficiency of the histidine-rich epidermal protein filaggrin (FLG) ... more Genetic deficiency or haploinsufficiency of the histidine-rich epidermal protein filaggrin (FLG) is associated with ichthyosis vulgaris (IV) and atopic dermatitis (AD). We investigated if the FLG genotype affects the microbiota composition of human skin and the cutaneous host response. FLG-deficient individuals had a low abundance of proteolytic Gram-positive anaerobic cocci (e.g. Finegoldia magna), which use peptides as nutrient sources. Furthermore, genes involved in histidine utilization were underrepresented in microbiota from FLG-deficient patients. An in vitro survival disadvantage of Finegoldia magna on FLGdeficient stratum corneum supported the in vivo findings. In co-cultures with primary human keratinocytes, Finegoldia magna induced a stronger antimicrobial peptide response than Staphylococcus aureus, whereas Staphylococcus aureus caused a stronger proinflammatory cytokine response than Finegoldia magna. Our data indicate that a common genetic defect can shape the cutaneous microbiome based on metabolic requirements of certain taxa, and may alter the host response to pathogens.

Journal of Leukocyte Biology, Nov 8, 2013

Intestinal M play an important role in maintaining gut homeostasis. However, little is known abou... more Intestinal M play an important role in maintaining gut homeostasis. However, little is known about these cells, their precursors, and their role in intestinal inflammation. Here, we characterize the CD14 ϩ mononuclear cell populations in intestinal mucosa and blood in patients with CD. Among the LP CD14 ϩ M, we identified three distinct HLA-DR ϩ-expressing subsets. Compared with uninflamed, inflamed mucosa contained a marked increase in the proportion of the CD14 hi HLA-DR dim cellular subset. This subset resembled the classical blood monocytes with low CD16, HLA-DR, and CX3CR1 expression. Classical monocytes migrated efficiently toward CCL2 and released the highest levels of MMP-1 and proinflammatory cytokines when stimulated with immune complexes or LPS. Our findings strongly suggest that it is the classical and not the intermediate or nonclassical monocytes that are the precursors to the dominating intestinal CD14 hi HLA-DR dim subset. This enhances our understanding of CD pathology and may provide new options in treatment.

Journal of Immunology, Aug 1, 2001

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitop... more The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L d 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.

Proceedings of the National Academy of Sciences of the United States of America, Jan 21, 2010

Biochemistry, Jul 21, 1999

Transient interactions between molecular chaperones and nascent polypeptide chains assist protein... more Transient interactions between molecular chaperones and nascent polypeptide chains assist protein folding in the endoplasmic reticulum. In an experimental setting that resembles the ER, we have used peptides as model substrates to identify and compare substrate specificities of ER-resident chaperones. The ER-located peptide transporter TAP was used to introduce peptides into the lumen of microsomes. In addition to PDI and gp96, previously identified as peptide-binding chaperones in the ER, we show that ERp72, calnexin, and grp170 interact with TAP-translocated peptides. The chaperones that have been identified can all bind peptide substrates that range from 8 to 40 amino acids in a manner independent of ATP. In addition, these chaperones exhibit broad and largely overlapping, however not identical, substrate selectivities. Our data indicate that peptide translocation into microsomes via TAP can be used as a method to monitor substrate selectivities of ER-resident chaperones. The implications of the observed preferences for chaperone-substrate interactions and for chaperones applied as vehicles in peptide-based vaccination strategies will be discussed.

Immunity, Apr 1, 2011

It is well established that natural killer (NK) cells confer resistance to many viral diseases, b... more It is well established that natural killer (NK) cells confer resistance to many viral diseases, but in only a few instances the molecular mechanisms whereby NK cells recognize virus-infected cells are known. Here we show that CD94, a molecule preferentially expressed by NK cells, is essential for the resistance of C57BL/6 mice to mousepox, a disease caused by the Orthopoxvirus ectromelia virus. Ectromelia virusinfected cells expressing the major histocompatibility complex (MHC) class Ib molecule Qa-1 b are specifically recognized by the activating receptor formed by CD94 and NKG2E. Because CD94-NKG2 receptors and their ligands are highly conserved in rodents and humans, a similar mechanism may exist during human infections with the smallpox and monkeypox viruses, which are highly homologous to ectromelia virus.

Biochemical and Biophysical Research Communications, Apr 1, 2003

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imid... more We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-ylethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic b-cells by stimulation of Ca 2þ-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074stimulated exocytosis was antagonised by the cytoplasmic phospholipase A 2 (cPLA 2) inhibitors ACA and AACOCF 3 and in cells treated with antisense oligonucleotide against cPLA 2 a. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF 3 , and cPLA 2 a antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA 2 activity. We propose that cPLA 2 a plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting b-cells.

Skin Research and Technology, Nov 1, 2016

Background/Purpose: FibroTx Transdermal Analyses Patch (TAP) is a novel technology for non-invasi... more Background/Purpose: FibroTx Transdermal Analyses Patch (TAP) is a novel technology for non-invasive measurements of protein biomarkers on the skin surface, in vivo. The aim of this study was to explore the potential of TAP in detecting skin surface biomarkers following mild perturbations, in vivo, using two experimental models: tape stripping, mimicking acute barrier disruption, and histamine iontophoresis, mimicking acute and local inflammation at minimal skin barrier insult. Methods: Tape stripping and histamine iontophoresis were performed in two separate experiments on the volar forearm of healthy volunteers (n = 27 and n = 10, respectively). Biomarker levels were assessed with TAP at baseline and up to 72 h after stimulation. Functional (transepidermal water loss-TEWL-and a* value) and morphological (confocal reflectance microscopy-RCM) assessments were added in the tape stripping and histamine iontophoresis experiments, respectively. Results: Cytokines IL-1a and IL-1RA and the antimicrobial peptide hBD-1 showed distinct dynamics, despite substantial inter-individual variation in levels, with an increase following tape stripping and a decrease following histamine iontophoresis. These dynamics could be related to the assessments made by TEWL and RCM. In the tape stripping experiment, additional biomarkers could be detected. Conclusion: TAP measurements, especially IL-1a, IL-1RA, and hBD-1, from the skin surface were sensitive enough for monitoring dynamic changes in the skin in the two models of skin perturbation. We conclude that TAP holds promise for non-invasively unraveling the dynamics of processes related to skin perturbation and repair.

Blood, Nov 16, 2007

In patients with acute myeloid leukemia (AML), haplo-identical stem cell transplantation (SCT) ca... more In patients with acute myeloid leukemia (AML), haplo-identical stem cell transplantation (SCT) can lead to expansion and activation of Killer Immunoglobulin-like Receptor (KIR)-HLA class I mismatched NK cells, resulting in reduced rates of leukemia relapse and no graft-versus-host disease (Ruggeri et al. Science 2002). However, this SCT is not available to the majority of AML patients who are elderly. To explore the feasibility of achieving similar NK-mediated anti-leukemia activity by a pharmacological approach, we generated fully human anti-KIR mAbs that block the interactions of inhibitory KIR2DL receptors with their HLA-C ligands, thereby enhancing NK activity. Here we describe one such therapeutic candidate anti-KIR mAb, designated 1-7F9. As distinct HLA-C allotypes are recognized by KIR2DL1 or −2/3, only mAbs that cross-react with these KIRs would be expected to work in the entire population. Hence, 1-7F9 was initially selected based on its ability to bind soluble, recombinant KIR2L1, −2 and −3. By Biacore analysis, the bivalent affinities for KIR2DL1 and −3 were 0.43 × 10−9 M and 0.025 × 10−9 M, respectively. In experimental systems and in normal human blood, 1-7F9 bound KIR2DL1, −2 and −3, and −2DS1 and −2, but not to KIR2DS3 or −4. 1-7F9 dose-dependently inhibited the binding of soluble KIR2DL1-Fc to cell surface HLA-Cw4. 1-7F9 augmented the lysis of 721.221-Cw4 B-EBV cells by an NK cell line transfected with KIR2DL1 (YTS-2DL1) from 5% lysis in absence of mAb to a maximal 55% lysis at 5 ug/ml of mAb, but did not affect lysis by KIR-negative NK cells. Lysis of PHA-stimulated blasts and primary AML blasts by autologous IL-2 activated NK cells (E:T=6:1) was 10 and 15%, respectively, in absence of mAb vs 80% and 55% in presence of 1-7F9. Incubation of IL-2 activated blood mononuclear cells with 1-7F9 resulted in expression of the activation marker CD107 on about 10% of KIR2D-positive NK cells, which increased to 20% upon addition of HLA-C-positive B-EBV targets, suggesting that 1-7F9 preferentially induces activation of NK cells in presence of transformed cells. The isotype of 1-7F9 is IgG4; accordingly, it did not cause depletion of KIR positive cells in vitro or in vivo in KIR-transgenic mice despite long-lived KIR-occupancy. As KIR are not found in mice, in vivo activity was tested in a NOD-SCID mouse model where inoculation of in vitro-expanded NK cells (80% of NK cells KIR2D-positive) and autologous human B-EBV cells (E:T=1:3) resulted in death of all mice by day 26. A single injection of 1-7F9 (125 ug/mouse) resulted in long-term survival, with 100% of treated mice alive beyond day 60; in contrast, 60 ug/mouse of the mAb was ineffective. Similarly, ex vivo pre-incubation of NK cells with 1-7F9 (37,3 ug/106 NK cells) prior to inoculation in mice resulted in elimination of the autologous transformed B cells in vivo and survival of 100% of the treated animals. These data show that 1-7F9 augments NK-mediated tumor killing in vitro and in vivo, and that it exhibits long-lived KIR binding in vivo, providing a preclinical basis for initiating phase 1 clinical trials with the mAb.

European Journal of Immunology, Sep 1, 1997

The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (... more The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (TAP) associates with peptides in the cytosol and translocates these into the ER lumen. Here, MHC class I molecules bind a subset of these peptides and the remainder is either removed or degraded, or may be retained in the ER in association with other proteins. We have visualized peptide-binding proteins in the ER using radioactive peptides with a photoreactive group. Besides TAP, two proteins were identified as gp96 and protein disulfide isomerase (PDI). Calreticulin, previously found in complex with TAP, only binds glycosylated peptides. In addition, two as yet unidentified, ER luminal glycoproteins (gp120 and gp170) were visualized. The effects of peptide size and sequence on binding to the ER-resident proteins were studied by using partially degenerated peptides with photoreactive side chains. All identified proteins were able to bind peptides within the size range of peptides translocated by TAP, from 8 to more than 20 amino acids. Whereas PDI associated with all peptides tested, gp96 and gp120 showed a clear sequence preference for non-charged amino acids at positions 2 and 9 in 9mer peptides. Thus various ER proteins, other than the MHC class I heterodimer and TAP, are able to interact with peptides albeit with a different substrate selectivity.

Blood, Sep 24, 2009

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK... more Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1,-2, and-3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cellmediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody. (Blood.

OncoImmunology, Apr 8, 2015

Annals of the Rheumatic Diseases, 2013

Conclusions: Our data indicate that mice deficient in mPGES-1 do not respond to vagus-mediated im... more Conclusions: Our data indicate that mice deficient in mPGES-1 do not respond to vagus-mediated immunosuppression. Moreover, we show immunosuppressant effects of PGE 2 on TNF release. These results implicate an involvement of PGE 2 as an important mediator in the cholinergic anti-inflammatory pathway, and provide support for further investigations regarding the interaction between cholinergic and prostaglandin systems.

Annals of the Rheumatic Diseases, 2014

ACR20, 50 and 70 response, remission (rem; defined as Clinical Disease Activity Index [CDAI] ≤2.8... more ACR20, 50 and 70 response, remission (rem; defined as Clinical Disease Activity Index [CDAI] ≤2.8, Simplified Disease Activity Index [SDAI] ≤3.3, Boolean score ≤1), low disease activity (LDA; CDAI ≤10, SDAI ≤11) or improvement in physical function (Health Assessment Questionnaire-Disability Index [HAQ-DI] >0.3) were assessed by disease duration and treatment subgroups. Results: 646 pts were randomized and treated with SC ABA (n=318) or ADA (n=328) on background MTX (71 and 70 pts with ≤6 months' disease duration; 247 and 258 pts with >6 months' disease duration). Baseline characteristics were balanced between the disease duration subgroups, with the exception of a higher percentage of males among pts with ≤6 months' disease duration treated with ADA compared with the other subgroups. Among pts with ≤6 months' disease duration, 23.9% (SC ABA) and 30.0% (ADA) of pts discontinued study treatment; 19.8% (SC ABA) and 24.0% (ADA) of pts with >6 months' disease duration discontinued. Clinical responses by disease duration at Years 1 (AMPLE primary endpoint) and 2 are summarized in the table. Conclusions: In contrast to what has been shown with DMARDs, data from this post hoc analysis of the AMPLE trial show that pts treated with effective biologic DMARDs (SC abatacept or adalimumab), whether treated early in the course of disease (≤6 months) or later, achieved comparable responses across a range of clinical measures.

Annals of the Rheumatic Diseases, 2013

ABSTRACT Background CD94/NKG2A+ CD56bright Natural Killer (NK) cells represent the majority of NK... more ABSTRACT Background CD94/NKG2A+ CD56bright Natural Killer (NK) cells represent the majority of NK cells in synovial fluid (SF) of Rheumatoid Arthritis (RA) patients and were proposed to exert an immunoregulatory role [1]. Prophylactic treatment with anti-NKG2A antibody fragment F(ab)’2 in murine collagen-induced arthritis reduced disease development by activating NK cells through blockade of its inhibitory receptor CD94/NKG2A [2]. Novo Nordisk A/S has generated an antagonistic, humanized anti-human NKG2A monoclonal antibody NNC141-0100 that blocks interaction between CD94/NKG2A and its ligand HLA-E. Objectives The aim of this study was to characterize the binding specificity of NNC141-0100 and the expression pattern of its target, the CD94/NKG2A receptors, in peripheral blood (PB), SF and/or synovial tissue from RA patients and healthy donors (HD). Methods CD94/NKG2A and HLA-E expression was investigated using flow cytometry, immunohistochemistry, digital image analysis and double-immunofluorescence (DIF) staining. Results In PB and SF from HD or RA patients, NNC141-0100 binding correlated with expression of CD94/NKG2A as detected by another anti-NKG2A mAb, and was restricted to subsets of NK and T cells. We observed a similar distribution of CD94/NKG2A on PB lymphocytes from HD and RA patients where ∼50% of NK cells expressed CD94/NKG2A. In contrast, the majority (>90%) of NK cells in RA SF were CD94/NKG2A+, confirming the previously reported disease-associated accumulation of CD94/NKG2A+ NK cells in RA SF [1]. CD94/NKG2A+ distribution on T cells was similar in PB from HD and RA patients and in RA SF (∼3%). In RA synovial tissue CD94/NKG2A was expressed by the majority of NK cells and a small subset of T cells. CD94/NKG2A+ cells were predominantly localized in lymphoid aggregates, but also present throughout RA synovium, whereas CD94/NKG2A+ cells were absent in synovium from HD. The CD94/NKG2A+ ligand, HLA-E, was detected on all infiltrating leukocytes in RA SF and synovium, at levels at least comparable to PB leucocytes from HD. Moreover, HLA-E was expressed by resident cells such as synoviocytes and endothelial cells in RA synovium. Conclusions These data demonstrate that CD94/NKG2A and its ligand HLA-E are expressed at sites of inflammation in RA. NNC141-0100 specifically binds to CD94/NKG2A+ NK cells accumulating in inflamed joints of RA patients, thus treatment with NNC141-0100 may promote the elimination of activated pro-inflammatory cells and suppress inflammation in RA patients. NNC141-0100 is currently being developed for the treatment of RA. Disclosure of Interest V. Pascal Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, Y. Sundström Grant/Research support from: Novo Nordisk A/S, A. Fasth Grant/Research support from: Novo Nordisk A/S, V. Malmström Grant/Research support from: Novo Nordisk A/S, L. Berg Grant/Research support from: Novo Nordisk A/S, P. Kvist Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, P. Spee Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, E. Galsgaard Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S

Annals of the Rheumatic Diseases, 2014

Methods: A549 cells were induced with IL-1b and treated with mPGES-1 inhibitor (compound III) or ... more Methods: A549 cells were induced with IL-1b and treated with mPGES-1 inhibitor (compound III) or COX-2 inhibitor (NS398). Eicosanoid profiles were analyzed using LC-MS/MS and protein profiles were analyzed using mass spectrometry based proteomics. FA composition of total lipids was determined using gas chromatography with flame ionization detector and sphingolipids were analysed by LC-MS/MS. Results: Prostanoid profiles in supernatants from the IL-1b-induced cells treated with the COX-2 or mPGES-1 inhibitors were significantly different suggesting diverse effects of the inhibitors on downstream pathways. Proteomic analysis of A549 cells identified the reduction of SCD levels in response to treatment with mPGES-1 or COX-2 inhibitors, as well as the suppression of the levels of a number of proteins involved in metabolism of fatty acids and sphingolipids. Conclusions: The results suggest that down-regulation of COX-2/mPGES-1/PGE2 axis affects the lipid metabolism in A549 cells. These effects have important implications regarding potential consequences of pharmacologic mPGES-1 inhibition.

Proceedings of the National Academy of Sciences of the United States of America, Aug 22, 2011

Acta dermato-venereologica, 2017

Interleukin-1α (IL-1α) and its receptor antagonist IL-1RA play a pivotal role in skin homeostasis... more Interleukin-1α (IL-1α) and its receptor antagonist IL-1RA play a pivotal role in skin homeostasis and disease. Although the use of biopsies to sample these cytokines from human skin is widely employed in dermatological practice, knowledge about less invasive, in vivo sampling methods is scarce. The aim of this study was to provide an overview of such methods by systematically reviewing studies in Medline, EMBASE, Web of Science and Cochrane Library using combinations of the terms "IL-1α", IL-1RA", "skin", "human", including all possible synonyms. Quality was assessed using the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) checklist. The search, performed on 14 October 2016, revealed 10 different sampling methods, with varying degrees of invasiveness and wide application spectrum, including assessment of both normal and diseased skin, from several body sites. The possibility to sample quantifiable amounts of cytokines from human skin with no or minimal discomfort holds promise for linking clinical outcomes to molecular profiles of skin inflammation.

Journal of Infectious Diseases and Therapy, Nov 2, 2016

Journal of Investigative Dermatology, Sep 1, 2016

Genetic deficiency or haploinsufficiency of the histidine-rich epidermal protein filaggrin (FLG) ... more Genetic deficiency or haploinsufficiency of the histidine-rich epidermal protein filaggrin (FLG) is associated with ichthyosis vulgaris (IV) and atopic dermatitis (AD). We investigated if the FLG genotype affects the microbiota composition of human skin and the cutaneous host response. FLG-deficient individuals had a low abundance of proteolytic Gram-positive anaerobic cocci (e.g. Finegoldia magna), which use peptides as nutrient sources. Furthermore, genes involved in histidine utilization were underrepresented in microbiota from FLG-deficient patients. An in vitro survival disadvantage of Finegoldia magna on FLGdeficient stratum corneum supported the in vivo findings. In co-cultures with primary human keratinocytes, Finegoldia magna induced a stronger antimicrobial peptide response than Staphylococcus aureus, whereas Staphylococcus aureus caused a stronger proinflammatory cytokine response than Finegoldia magna. Our data indicate that a common genetic defect can shape the cutaneous microbiome based on metabolic requirements of certain taxa, and may alter the host response to pathogens.

Journal of Leukocyte Biology, Nov 8, 2013

Intestinal M play an important role in maintaining gut homeostasis. However, little is known abou... more Intestinal M play an important role in maintaining gut homeostasis. However, little is known about these cells, their precursors, and their role in intestinal inflammation. Here, we characterize the CD14 ϩ mononuclear cell populations in intestinal mucosa and blood in patients with CD. Among the LP CD14 ϩ M, we identified three distinct HLA-DR ϩ-expressing subsets. Compared with uninflamed, inflamed mucosa contained a marked increase in the proportion of the CD14 hi HLA-DR dim cellular subset. This subset resembled the classical blood monocytes with low CD16, HLA-DR, and CX3CR1 expression. Classical monocytes migrated efficiently toward CCL2 and released the highest levels of MMP-1 and proinflammatory cytokines when stimulated with immune complexes or LPS. Our findings strongly suggest that it is the classical and not the intermediate or nonclassical monocytes that are the precursors to the dominating intestinal CD14 hi HLA-DR dim subset. This enhances our understanding of CD pathology and may provide new options in treatment.

Journal of Immunology, Aug 1, 2001

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitop... more The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-L d 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.

Proceedings of the National Academy of Sciences of the United States of America, Jan 21, 2010

Biochemistry, Jul 21, 1999

Transient interactions between molecular chaperones and nascent polypeptide chains assist protein... more Transient interactions between molecular chaperones and nascent polypeptide chains assist protein folding in the endoplasmic reticulum. In an experimental setting that resembles the ER, we have used peptides as model substrates to identify and compare substrate specificities of ER-resident chaperones. The ER-located peptide transporter TAP was used to introduce peptides into the lumen of microsomes. In addition to PDI and gp96, previously identified as peptide-binding chaperones in the ER, we show that ERp72, calnexin, and grp170 interact with TAP-translocated peptides. The chaperones that have been identified can all bind peptide substrates that range from 8 to 40 amino acids in a manner independent of ATP. In addition, these chaperones exhibit broad and largely overlapping, however not identical, substrate selectivities. Our data indicate that peptide translocation into microsomes via TAP can be used as a method to monitor substrate selectivities of ER-resident chaperones. The implications of the observed preferences for chaperone-substrate interactions and for chaperones applied as vehicles in peptide-based vaccination strategies will be discussed.

Immunity, Apr 1, 2011

It is well established that natural killer (NK) cells confer resistance to many viral diseases, b... more It is well established that natural killer (NK) cells confer resistance to many viral diseases, but in only a few instances the molecular mechanisms whereby NK cells recognize virus-infected cells are known. Here we show that CD94, a molecule preferentially expressed by NK cells, is essential for the resistance of C57BL/6 mice to mousepox, a disease caused by the Orthopoxvirus ectromelia virus. Ectromelia virusinfected cells expressing the major histocompatibility complex (MHC) class Ib molecule Qa-1 b are specifically recognized by the activating receptor formed by CD94 and NKG2E. Because CD94-NKG2 receptors and their ligands are highly conserved in rodents and humans, a similar mechanism may exist during human infections with the smallpox and monkeypox viruses, which are highly homologous to ectromelia virus.

Biochemical and Biophysical Research Communications, Apr 1, 2003

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imid... more We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-ylethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic b-cells by stimulation of Ca 2þ-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074stimulated exocytosis was antagonised by the cytoplasmic phospholipase A 2 (cPLA 2) inhibitors ACA and AACOCF 3 and in cells treated with antisense oligonucleotide against cPLA 2 a. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF 3 , and cPLA 2 a antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA 2 activity. We propose that cPLA 2 a plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting b-cells.

Skin Research and Technology, Nov 1, 2016

Background/Purpose: FibroTx Transdermal Analyses Patch (TAP) is a novel technology for non-invasi... more Background/Purpose: FibroTx Transdermal Analyses Patch (TAP) is a novel technology for non-invasive measurements of protein biomarkers on the skin surface, in vivo. The aim of this study was to explore the potential of TAP in detecting skin surface biomarkers following mild perturbations, in vivo, using two experimental models: tape stripping, mimicking acute barrier disruption, and histamine iontophoresis, mimicking acute and local inflammation at minimal skin barrier insult. Methods: Tape stripping and histamine iontophoresis were performed in two separate experiments on the volar forearm of healthy volunteers (n = 27 and n = 10, respectively). Biomarker levels were assessed with TAP at baseline and up to 72 h after stimulation. Functional (transepidermal water loss-TEWL-and a* value) and morphological (confocal reflectance microscopy-RCM) assessments were added in the tape stripping and histamine iontophoresis experiments, respectively. Results: Cytokines IL-1a and IL-1RA and the antimicrobial peptide hBD-1 showed distinct dynamics, despite substantial inter-individual variation in levels, with an increase following tape stripping and a decrease following histamine iontophoresis. These dynamics could be related to the assessments made by TEWL and RCM. In the tape stripping experiment, additional biomarkers could be detected. Conclusion: TAP measurements, especially IL-1a, IL-1RA, and hBD-1, from the skin surface were sensitive enough for monitoring dynamic changes in the skin in the two models of skin perturbation. We conclude that TAP holds promise for non-invasively unraveling the dynamics of processes related to skin perturbation and repair.

Blood, Nov 16, 2007

In patients with acute myeloid leukemia (AML), haplo-identical stem cell transplantation (SCT) ca... more In patients with acute myeloid leukemia (AML), haplo-identical stem cell transplantation (SCT) can lead to expansion and activation of Killer Immunoglobulin-like Receptor (KIR)-HLA class I mismatched NK cells, resulting in reduced rates of leukemia relapse and no graft-versus-host disease (Ruggeri et al. Science 2002). However, this SCT is not available to the majority of AML patients who are elderly. To explore the feasibility of achieving similar NK-mediated anti-leukemia activity by a pharmacological approach, we generated fully human anti-KIR mAbs that block the interactions of inhibitory KIR2DL receptors with their HLA-C ligands, thereby enhancing NK activity. Here we describe one such therapeutic candidate anti-KIR mAb, designated 1-7F9. As distinct HLA-C allotypes are recognized by KIR2DL1 or −2/3, only mAbs that cross-react with these KIRs would be expected to work in the entire population. Hence, 1-7F9 was initially selected based on its ability to bind soluble, recombinant KIR2L1, −2 and −3. By Biacore analysis, the bivalent affinities for KIR2DL1 and −3 were 0.43 × 10−9 M and 0.025 × 10−9 M, respectively. In experimental systems and in normal human blood, 1-7F9 bound KIR2DL1, −2 and −3, and −2DS1 and −2, but not to KIR2DS3 or −4. 1-7F9 dose-dependently inhibited the binding of soluble KIR2DL1-Fc to cell surface HLA-Cw4. 1-7F9 augmented the lysis of 721.221-Cw4 B-EBV cells by an NK cell line transfected with KIR2DL1 (YTS-2DL1) from 5% lysis in absence of mAb to a maximal 55% lysis at 5 ug/ml of mAb, but did not affect lysis by KIR-negative NK cells. Lysis of PHA-stimulated blasts and primary AML blasts by autologous IL-2 activated NK cells (E:T=6:1) was 10 and 15%, respectively, in absence of mAb vs 80% and 55% in presence of 1-7F9. Incubation of IL-2 activated blood mononuclear cells with 1-7F9 resulted in expression of the activation marker CD107 on about 10% of KIR2D-positive NK cells, which increased to 20% upon addition of HLA-C-positive B-EBV targets, suggesting that 1-7F9 preferentially induces activation of NK cells in presence of transformed cells. The isotype of 1-7F9 is IgG4; accordingly, it did not cause depletion of KIR positive cells in vitro or in vivo in KIR-transgenic mice despite long-lived KIR-occupancy. As KIR are not found in mice, in vivo activity was tested in a NOD-SCID mouse model where inoculation of in vitro-expanded NK cells (80% of NK cells KIR2D-positive) and autologous human B-EBV cells (E:T=1:3) resulted in death of all mice by day 26. A single injection of 1-7F9 (125 ug/mouse) resulted in long-term survival, with 100% of treated mice alive beyond day 60; in contrast, 60 ug/mouse of the mAb was ineffective. Similarly, ex vivo pre-incubation of NK cells with 1-7F9 (37,3 ug/106 NK cells) prior to inoculation in mice resulted in elimination of the autologous transformed B cells in vivo and survival of 100% of the treated animals. These data show that 1-7F9 augments NK-mediated tumor killing in vitro and in vivo, and that it exhibits long-lived KIR binding in vivo, providing a preclinical basis for initiating phase 1 clinical trials with the mAb.

European Journal of Immunology, Sep 1, 1997

The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (... more The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (TAP) associates with peptides in the cytosol and translocates these into the ER lumen. Here, MHC class I molecules bind a subset of these peptides and the remainder is either removed or degraded, or may be retained in the ER in association with other proteins. We have visualized peptide-binding proteins in the ER using radioactive peptides with a photoreactive group. Besides TAP, two proteins were identified as gp96 and protein disulfide isomerase (PDI). Calreticulin, previously found in complex with TAP, only binds glycosylated peptides. In addition, two as yet unidentified, ER luminal glycoproteins (gp120 and gp170) were visualized. The effects of peptide size and sequence on binding to the ER-resident proteins were studied by using partially degenerated peptides with photoreactive side chains. All identified proteins were able to bind peptides within the size range of peptides translocated by TAP, from 8 to more than 20 amino acids. Whereas PDI associated with all peptides tested, gp96 and gp120 showed a clear sequence preference for non-charged amino acids at positions 2 and 9 in 9mer peptides. Thus various ER proteins, other than the MHC class I heterodimer and TAP, are able to interact with peptides albeit with a different substrate selectivity.

Blood, Sep 24, 2009

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK... more Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1,-2, and-3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cellmediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody. (Blood.