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1.
Bioorg Med Chem ; 90: 117350, 2023 07 15.
Article En | MEDLINE | ID: mdl-37270903

To develop matrix metalloproteinase inhibitors (MMPIs) for both therapy and medicinal imaging by fluorescence-based techniques or positron-emission tomography (PET), a small library of eighteen N-substituted N-arylsulfonamido d-valines were synthesized and their potency to inhibit two gelatinases (MMP-2, and MMP-9), two collagenases (MMP-8, and MMP-13) and macrophage elastase (MMP-12) was determined in a Structure-Activity-Relation study with ({4-[3-(5-methylthiophen-2-yl)-1,2,4-oxadiazol-5-yl]phenyl}sulfonyl)-d-valine (1) as a lead. All compounds were shown to be more potent MMP-2/-9 inhibitors (nanomolar range) compared to other tested MMPs. This is a remarkable result considering that a carboxylic acid group is the zinc binding moiety. The compound with a terminal fluoropropyltriazole group at the furan ring (P1' substituent) was only four times less potent in inhibiting MMP-2 activity than the lead compound 1, making this compound a promising probe for PET application (after using a prosthetic group approach to introduce fluorine-18). Compounds with a TEG spacer and a terminal azide or even a fluorescein moiety at the sulfonylamide N atom (P2' substituent) were almost as active as the lead structure 1, making the latter derivative a suitable fluorescence imaging tool.


Matrix Metalloproteinase 2 , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinase Inhibitors/pharmacology , Structure-Activity Relationship , Valine , Carboxylic Acids
2.
Pharmaceuticals (Basel) ; 16(5)2023 Apr 27.
Article En | MEDLINE | ID: mdl-37242439

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 µM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 µM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 µM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

3.
Int J Mol Sci ; 24(7)2023 Mar 25.
Article En | MEDLINE | ID: mdl-37047188

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.


Myeloid-Derived Suppressor Cells , Neoplasms , Animals , Mice , Cell Line , Monocytes , Myeloid Cells , Myeloid-Derived Suppressor Cells/metabolism , Neoplasms/metabolism , Tumor Microenvironment
4.
FEBS J ; 290(4): 970-973, 2023 02.
Article En | MEDLINE | ID: mdl-36315610

So far one gene for Hv1 has been detected in studied species. The work presented by Chaves et al. in The FEBS Journal reported an 'Unexpected expansion of the voltage-gated proton channel family'. They searched for proton channel candidates and found three sequences in the genome of Aplysia californica (Ac), which were named AcHv1, AcHv2 and AcHv3. Based on electrophysiological experiments, AcHv1 and AcHv2 are voltage-gated channels. While AcHv1 behaves like Hv1 in other species, that is, it is voltage and pH-dependent, it can be inhibited by zinc and conducts protons outwardly, AcHv2 conducts protons inwards at symmetrical pH. AcHv3 constantly leaks protons, and its C-terminal part contains several cytoplasmic retention motifs. Through carefully designed and carried out electrophysiological experiments, Chaves et al. determined the biophysical parameters of all three proton channels, such as the voltage and the pH dependence, the threshold-voltage, the gating charge and the time constants of activation and inactivation. Comment on: https://doi.org/10.1111/febs.16617.


Ion Channel Gating , Protons , Ion Channel Gating/genetics , Ion Channels/metabolism , Zinc/metabolism
5.
Diabetologia ; 64(7): 1626-1641, 2021 07.
Article En | MEDLINE | ID: mdl-33912981

AIMS/HYPOTHESIS: We and others previously reported the presence of tertiary lymphoid organs (TLOs) in the pancreas of NOD mice, where they play a role in the development of type 1 diabetes. Our aims here are to investigate whether TLOs are present in the pancreas of individuals with type 1 diabetes and to characterise their distinctive features, in comparison with TLOs present in NOD mouse pancreases, in order to interpret their functional significance. METHODS: Using immunofluorescence confocal microscopy, we examined the extracellular matrix (ECM) and cellular constituents of pancreatic TLOs from individuals with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: at risk of diabetes; at/after diagnosis; and in the transplanted pancreas with recurrent diabetes. Comparisons were made with TLOs from 14-week-old NOD mice, which contain islets exhibiting mild to heavy leucocyte infiltration. We determined the frequency of the TLOs in human type 1diabetes with insulitis and investigated the presence of TLOs in relation to age of onset, disease duration and disease severity. RESULTS: TLOs were identified in preclinical and clinical settings of human type 1 diabetes. The main characteristics of these TLOs, including the cellular and ECM composition of reticular fibres (RFs), the presence of high endothelial venules and immune cell subtypes detected, were similar to those observed for TLOs from NOD mouse pancreases. Among 21 donors with clinical type 1 diabetes who exhibited insulitis, 12 had TLOs and had developed disease at younger age compared with those lacking TLOs. Compartmentalised TLOs with distinct T cell and B cell zones were detected in donors with short disease duration. Overall, TLOs were mainly associated with insulin-containing islets and their frequency decreased with increasing severity of beta cell loss. Parallel studies in NOD mice further revealed some differences in so far as regulatory T cells were essentially absent from human pancreatic TLOs and CCL21 was not associated with RFs. CONCLUSIONS/INTERPRETATION: We demonstrate a novel feature of pancreas pathology in type 1 diabetes. TLOs represent a potential site of autoreactive effector T cell generation in islet autoimmunity and our data from mouse and human tissues suggest that they disappear once the destructive process has run its course. Thus, TLOs may be important for type 1 diabetes progression.


Diabetes Mellitus, Type 1/pathology , Tertiary Lymphoid Structures/pathology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Autoantibodies/analysis , Autoantibodies/blood , Autoimmunity/physiology , Child , Child, Preschool , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/immunology , Female , Humans , Islets of Langerhans/pathology , Male , Mice , Mice, Inbred NOD , Microscopy, Fluorescence , Middle Aged , Pancreas/pathology , Tertiary Lymphoid Structures/blood , Tertiary Lymphoid Structures/immunology , Young Adult
6.
Diabetes ; 68(10): 1990-2003, 2019 10.
Article En | MEDLINE | ID: mdl-31371518

Tumors induce tolerance toward their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, nonobese diabetic (NOD) mice express CCL21 in pancreatic ß-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes. Ins2-CCL21 mice develop TLOs by 4 weeks of age, consisting of naive CD4+ T cells compartmentalized within networks of CD45-gp38+CD31- fibroblastic reticular cell (FRC)-like cells. Importantly, 12-week-old Ins2-CCL21 TLOs contained FRC-like cells with higher contractility, regulatory, and anti-inflammatory properties and enhanced expression of ß-cell autoantigens compared with nontransgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and a higher proportion of regulatory T cells in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for the regulation of autoimmunity, and although the effect is systemic, the induction is mediated locally likely by lymphocyte trafficking through TLOs. Overall, our findings suggest that CCL21 promotes TLOs that differ from inflammatory TLOs found in type 1 diabetic islets in that they resemble lymph nodes, contain FRC-like cells expressing ß-cell autoantigens, and are able to induce systemic and antigen-specific tolerance leading to diabetes prevention.


Chemokine CCL21/metabolism , Diabetes Mellitus, Type 1/metabolism , Insulin-Secreting Cells/metabolism , Pancreas/metabolism , Stromal Cells/metabolism , Animals , Mice , Mice, Inbred NOD , Mice, Transgenic
7.
Bioconjug Chem ; 29(11): 3715-3725, 2018 11 21.
Article En | MEDLINE | ID: mdl-30277751

Matrix metalloproteinases (MMPs) are emerging as pivotal fine-tuners of cell function in tissue homeostasis and in various pathologies, in particular inflammation. In vivo monitoring of the activity of specific MMPs, therefore, provides high potential for assessing disease progression and tissue function, and manipulation of MMP activity in tissues and whole organisms may further provide a mode of controlling pathological processes. We describe here the synthesis of novel fluorinated and nonfluorinated analogues of a secondary sulfonamide-based lead structure, compound 2, and test their efficacy as in vivo inhibitors and tracers of the gelatinases, MMP-2 and MMP-9. Using a murine neuroinflammatory model, we show that compound 2 is a highly effective in vivo inhibitor of both MMP-2 and MMP-9 activity with little or no adverse effects even after long-term daily oral administration. A fluorescein-labeled derivative compound 17 shows direct binding to activated gelatinases surrounding inflammatory cuffs in the neuroinflammation model and to pancreatic ß-cells in the islets of Langerhans, colocalizing with MMP-2 and MMP-9 activity as detected using in situ zymography techniques. These results demonstrate that compound 2 derivatives have potential as in vivo imaging tools and for future development for specific MMP-2 versus MMP-9 probes. Our chemical modifications mainly target the residues directed toward the S1' and S2' pockets and, thereby, provide new information on the structure-activity relationships of this inhibitor type.


Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase Inhibitors/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , Animals , Cell Line , Female , Halogenation , Humans , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/enzymology , Insulin-Secreting Cells/metabolism , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase Inhibitors/adverse effects , Matrix Metalloproteinase Inhibitors/chemical synthesis , Mice, Inbred C57BL , Molecular Docking Simulation , Structure-Activity Relationship , Sulfonamides/adverse effects , Sulfonamides/chemical synthesis
8.
J Neuroinflammation ; 15(1): 236, 2018 Aug 22.
Article En | MEDLINE | ID: mdl-30134924

BACKGROUND: Very late antigen 4 (VLA-4; integrin α4ß1) is critical for transmigration of T helper (TH) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human TH17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation. METHODS: Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays. RESULTS: Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4-/-), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, ß1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue. CONCLUSIONS: Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.


CD146 Antigen/metabolism , Choroid Plexus/pathology , Encephalomyelitis, Autoimmune, Experimental/pathology , T-Lymphocytes/drug effects , Animals , Antibodies/therapeutic use , CD146 Antigen/immunology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Central Nervous System/pathology , Choroid Plexus/diagnostic imaging , Choroid Plexus/metabolism , Cytokines/metabolism , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Endothelial Cells/drug effects , Freund's Adjuvant/toxicity , Humans , Integrin alpha4beta1/genetics , Integrin alpha4beta1/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myelin-Oligodendrocyte Glycoprotein/toxicity , Peptide Fragments/toxicity , Protein Kinases/genetics , Protein Kinases/metabolism
9.
Int J Med Microbiol ; 308(7): 761-769, 2018 Oct.
Article En | MEDLINE | ID: mdl-29843979

Staphylococcus aureus is the most frequent pathogen causing diabetic foot infections. Here, we investigated the degree of bacterial virulence required to establish invasive tissue infections in diabetic organisms. Staphylococcal isolates from diabetic and non-diabetic foot ulcers were tested for their virulence in in vitro functional assays of host cell invasion and cytotoxicity. Isolates from diabetes mellitus type I/II patients exhibited less virulence than isolates from non-diabetic patients, but were nevertheless able to establish severe infections. In some cases, non-invasive isolates were detected deep within diabetic wounds, even though the strains were non-pathogenic in cell culture models. Testing of defined isolates in murine footpad injection models revealed that both low- and high-virulent bacterial strains persisted in higher numbers in diabetic compared to non-diabetic hosts, suggesting that hyperglycemia favors bacterial survival. Additionally, the bacterial load was higher in NOD mice, which have a compromised immune system, compared to C57Bl/6 mice. Our results reveal that high as well as low-virulent staphylococcal strains are able to cause soft tissue infections and to persist in diabetic humans and mice, suggesting a reason for the frequent and endangering infections in patients with diabetes.


Bacterial Load , Diabetes Mellitus, Experimental/pathology , Diabetic Foot/microbiology , Soft Tissue Infections/microbiology , Staphylococcus aureus/pathogenicity , Adult , Aged , Animals , Cells, Cultured , Human Umbilical Vein Endothelial Cells , Humans , Hyperglycemia/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Middle Aged , Staphylococcus aureus/isolation & purification , Streptozocin , Virulence
10.
Matrix Biol ; 67: 47-62, 2018 04.
Article En | MEDLINE | ID: mdl-29360499

Hematopoietic stem and progenitor cell (HSPC) functions are regulated by a specialized microenvironment in the bone marrow - the hematopoietic stem cell niche - of which the extracellular matrix (ECM) is an integral component. We describe here the localization of ECM molecules, in particular the laminin α4, α3 and α5 containing isoforms in the bone marrow. Laminin 421 (composed of laminin α4, ß2, γ1 chains) is identified as a major component of the bone marrow ECM, occurring abundantly surrounding venous sinuses and in a specialized reticular fiber network of the intersinusoidal spaces of murine bone marrow (BM) in close association with HSPC. Bone marrow from Lama4-/- mice is significantly less efficient in reconstituting the hematopoietic system of irradiated wildtype (WT) recipients in competitive bone marrow transplantation assays and shows reduced colony formation in vitro. This is partially due to retention of Lin-c-kit+Sca-1+CD48- long-term and short-term hematopoietic stem cells (LT-HSC/ST-HSC) in the G0 phase of the cell cycle in Lama4-/- bone marrow and hence a more quiescent phenotype. In addition, the extravasation of WT BM cells into Lama4-/- bone marrow is impaired, influencing the recirculation of HSPC. Our data suggest that these effects are mediated by a compensatory expression of laminin α5 containing isoforms (laminin 521/522) in Lama4-/- bone marrow. Collectively, these intrinsic and extrinsic effects lead to reduced HSPC numbers in Lama4-/- bone marrow and reduced hematopoietic potential.


Bone Marrow/metabolism , Hematopoietic Stem Cells/cytology , Laminin/metabolism , Animals , Cell Cycle , Cell Movement , Extracellular Matrix/metabolism , Hematopoietic Stem Cells/metabolism , Laminin/genetics , Mice , Stem Cell Niche
11.
Sci Transl Med ; 8(364): 364ra152, 2016 11 09.
Article En | MEDLINE | ID: mdl-27831901

The enzymes gelatinase A/matrix metalloproteinase-2 (MMP-2) and gelatinase B/MMP-9 are essential for induction of neuroinflammatory symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS); in the absence of these enzymes, the disease does not develop. We therefore investigated the cellular sources and relative contributions of MMP-2 and MMP-9 to disease at early stages of EAE induction. We demonstrated that MMP-9 from an immune cell source is required in EAE for initial infiltration of leukocytes into the central nervous system and that MMP-9 activity is a reliable marker of leukocyte penetration of the blood-brain barrier. We then developed a molecular imaging method to visualize MMP activity in the brain using fluorescent- and radioactive-labeled MMP inhibitors (MMPis) in EAE animals and used the radioactive MMP ligand for positron emission tomography (PET) imaging of MMP activity in patients with MS. In contrast to traditional T1-gadolinium contrast-enhanced MRI, MMPi-PET enabled tracking of MMP activity as a unique feature of early lesions and ongoing leukocyte infiltration. MMPi-PET therefore allows monitoring of the early steps of MS development and provides a sensitive, noninvasive means of following lesion formation and resolution in murine EAE and human MS.


Blood-Brain Barrier/metabolism , Leukocytes/cytology , Matrix Metalloproteinases/metabolism , Multiple Sclerosis/genetics , Multiple Sclerosis/physiopathology , Animals , Brain/metabolism , Brain/pathology , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/pathology , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immune System , Inflammation , Ligands , Magnetic Resonance Imaging , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiple Sclerosis/enzymology , Positron-Emission Tomography , Spinal Cord/metabolism
12.
J Exp Med ; 212(10): 1529-49, 2015 Sep 21.
Article En | MEDLINE | ID: mdl-26347470

Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood-brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-κB and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-κB-independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1-NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP.


Brain/blood supply , Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Kinase Kinases/metabolism , Animals , Blood-Brain Barrier/metabolism , Brain/metabolism , Cerebrovascular Circulation/genetics , Endothelial Cells/metabolism , Endothelial Cells/pathology , Epilepsy/genetics , Female , I-kappa B Kinase/metabolism , Incontinentia Pigmenti/metabolism , Incontinentia Pigmenti/pathology , Intracellular Signaling Peptides and Proteins/genetics , MAP Kinase Kinase Kinases/genetics , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Occludin/metabolism , TNF Receptor-Associated Factor 6/metabolism , Transcription Factor RelA/metabolism
13.
Neural Regen Res ; 10(6): 866-9, 2015 Jun.
Article En | MEDLINE | ID: mdl-26199591

The extracellular matrix (ECM) performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2) in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth.

14.
Cell Rep ; 10(7): 1040-54, 2015 Feb 24.
Article En | MEDLINE | ID: mdl-25704809

Although chemokines are sufficient for chemotaxis of various cells, increasing evidence exists for their fine-tuning by selective proteolytic processing. Using a model of immune cell chemotaxis into the CNS (experimental autoimmune encephalomyelitis [EAE]) that permits precise localization of immigrating leukocytes at the blood-brain barrier, we show that, whereas chemokines are required for leukocyte migration into the CNS, additional MMP-2/9 activities specifically at the border of the CNS parenchyma strongly enhance this transmigration process. Cytokines derived from infiltrating leukocytes regulate MMP-2/9 activity at the parenchymal border, which in turn promotes astrocyte secretion of chemokines and differentially modulates the activity of different chemokines at the CNS border, thereby promoting leukocyte migration out of the cuff. Hence, cytokines, chemokines, and cytokine-induced MMP-2/9 activity specifically at the inflammatory border collectively act to accelerate leukocyte chemotaxis across the parenchymal border.


Blood-Brain Barrier/metabolism , Chemokines/metabolism , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Animals , Astrocytes/cytology , Astrocytes/drug effects , Astrocytes/metabolism , Cell Movement/drug effects , Cells, Cultured , Central Nervous System/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Interferon-gamma/pharmacology , Interleukin-17/pharmacology , Leukocytes/cytology , Leukocytes/metabolism , Matrix Metalloproteinase 2/deficiency , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/deficiency , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/pharmacology
15.
Curr Diab Rep ; 14(12): 552, 2014 Dec.
Article En | MEDLINE | ID: mdl-25344787

Type 1 diabetes (T1D) results from progressive immune cell-mediated destruction of pancreatic ß cells. As immune cells migrate into the islets, they pass through the extracellular matrix (ECM). This ECM is composed of different macromolecules localized to different compartments within and surrounding islets; however, the involvement of this ECM in the development of human T1D is not well understood. Here, we summarize our recent findings from human and mouse studies illustrating how specific components of the islet ECM that constitute basement membranes and interstitial matrix of the islets, and surprisingly, the intracellular composition of islet ß cells themselves, are significantly altered during the pathogenesis of T1D. Our focus is on the ECM molecules laminins, collagens, heparan sulfate/heparan sulfate proteoglycans, and hyaluronan, as well as on the enzymes that degrade these ECM components. We propose that islet and lymphoid tissue ECM composition and organization are critical to promoting immune cell activation, islet invasion, and destruction of islet ß cells in T1D.


Diabetes Mellitus, Type 1/etiology , Extracellular Matrix/metabolism , Animals , Basement Membrane/metabolism , Humans , Pancreas/metabolism , Pancreas/pathology , Proteoglycans/metabolism
16.
J Cell Sci ; 127(Pt 15): 3240-56, 2014 Aug 01.
Article En | MEDLINE | ID: mdl-24895400

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-ß1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2(-/-) relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2(-/-) fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.


Extracellular Matrix/metabolism , Matrilin Proteins/metabolism , Muscles/physiology , Myoblasts/physiology , Necrosis/therapy , Animals , Apoptosis/genetics , Cell Line , Cell Proliferation/genetics , Elapid Venoms/administration & dosage , Humans , Matrilin Proteins/genetics , Mice , Mice, Knockout , Muscle Development/genetics , Muscles/pathology , Necrosis/chemically induced , Rats , Rats, Wistar , Regeneration/genetics , Time Factors
18.
Diabetes ; 62(2): 531-42, 2013 Feb.
Article En | MEDLINE | ID: mdl-23139348

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. Protease- and protease inhibitor-specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.


Basement Membrane/immunology , Chemotaxis, Leukocyte/immunology , Diabetes Mellitus, Type 1/immunology , Extracellular Matrix/immunology , Islets of Langerhans/immunology , Animals , Cathepsin C/analysis , Cathepsin W/analysis , Cathepsins/analysis , Humans , Inflammation/metabolism , Islets of Langerhans/pathology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Inbred NOD , Protein Array Analysis , Proteinase Inhibitory Proteins, Secretory/analysis
19.
J Immunol ; 188(7): 3138-49, 2012 Apr 01.
Article En | MEDLINE | ID: mdl-22371394

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αß type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αß NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αß NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.


Apoptosis/immunology , Diabetes Mellitus, Type 1/immunology , Natural Killer T-Cells/immunology , T-Lymphocyte Subsets/immunology , Adoptive Transfer , Animals , Antigens, CD1d/immunology , B7-H1 Antigen/physiology , CD4-Positive T-Lymphocytes/classification , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/transplantation , Diabetes Mellitus, Type 1/prevention & control , Disease Models, Animal , Immunologic Memory , Inducible T-Cell Co-Stimulator Protein/physiology , Lymph Nodes/immunology , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Natural Killer T-Cells/classification , Natural Killer T-Cells/transplantation , Pancreas/immunology , Pancreatitis/prevention & control , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Specific Pathogen-Free Organisms , T-Lymphocyte Subsets/transplantation , T-Lymphocytes, Regulatory/immunology
20.
Int J Cancer ; 131(4): E425-36, 2012 Aug 15.
Article En | MEDLINE | ID: mdl-21953247

Chlorite-oxidized oxyamylose (COAM), a glycosaminoglycan mimetic and potent antiviral agent, provided significant growth reduction of syngeneic murine B16-F1 melanoma tumors. A single early dose (100 µg, into the site of tumor cell inoculation) was sufficient to establish a persistent effect over 17 days (resected tumor volume of 78.3 mm(3) in COAM-treated mice compared to 755.2 mm(3) in the control cohort, i.e., 89.6% reduction of tumor volumes). COAM was a much better antitumoral agent than the polyanionic glycosaminoglycan heparin. COAM retained its antitumoral effect in lymphopenic mice, reinforcing the idea of myeloid cell involvement. Massive recruitment of myeloid cells into dermal air pouches in response to COAM and their increased presence in early-treated tumors indicated that mainly CD11b(+) GR-1(+) myeloid cells were attracted by COAM to exert antitumoral effects. Leukocyte chemotaxis was mediated by the chemokine system through the induction in B16-F1 cells of mouse granulocyte chemotactic protein-2/CXCL6 upon COAM treatment. Thus, COAM constitutes a novel tool to study the role of innate immune cells in the initial stages of tumor development and an example that innate immunostimulating glycosaminoglycan mimicry may be exploited therapeutically.


Amylose/analogs & derivatives , Chemokines/biosynthesis , Glycosaminoglycans/metabolism , Melanoma, Experimental/pathology , Molecular Mimicry , Amylose/pharmacology , Animals , Cell Division/drug effects , Flow Cytometry , Fluorescent Dyes , Male , Melanoma, Experimental/metabolism , Mice , Mice, Nude , Real-Time Polymerase Chain Reaction
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