Identification of a Novel Structural Class of H1 Inhibitors by Structure-Based Virtual Screening.

The human voltage-gated proton channel, hH1, is highly expressed in various cell types including macrophages, B lymphocytes, microglia, sperm cells and also in various cancer cells. Overexpression of H1 has been shown to promote tumor formation by highly metastatic cancer cells, and has been associated with neuroinflammatory diseases, immune response disorders and infertility, suggesting a potential use of hH1 inhibitors in numerous therapeutic areas. To identify compounds targeting this channel, we performed a structure-based virtual screening on an open structure of the human H1 channel.

Influence of N-arylsulfonamido d-valine N-substituents on the selectivity and potency of matrix metalloproteinase inhibitors.

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.

5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human H1 Channel.

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H) channel (H1, ≈ 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.

The Voltage-Gated Hv1 H Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

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).

New 'kids' on the voltage-gated proton channel block.

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'.

Identification and characterisation of tertiary lymphoid organs in human type 1 diabetes.

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.

CCL21 Expression in β-Cells Induces Antigen-Expressing Stromal Cell Networks in the Pancreas and Prevents Autoimmune Diabetes in Mice.

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 β-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes.

New in Vivo Compatible Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitors.

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.

Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus.

Background: Very late antigen 4 (VLA-4; integrin α4β1) is critical for transmigration of T helper (T) 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 T17 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.

Staphylococcus aureus requires less virulence to establish an infection in diabetic hosts.

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.

Bone marrow laminins influence hematopoietic stem and progenitor cell cycling and homing to the bone marrow.

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, β2, 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.

Imaging matrix metalloproteinase activity in multiple sclerosis as a specific marker of leukocyte penetration of the blood-brain barrier.

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.

Brain endothelial TAK1 and NEMO safeguard the neurovascular unit.

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.

Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues.

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.

Focal MMP-2 and MMP-9 activity at the blood-brain barrier promotes chemokine-induced leukocyte migration.

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.

Extracellular matrix components in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) results from progressive immune cell-mediated destruction of pancreatic β 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.

Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration.

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.

The peri-islet basement membrane, a barrier to infiltrating leukocytes in type 1 diabetes in mouse and human.

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.

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic β 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.

Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function.

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.

Role of the extracellular matrix in lymphocyte migration.

The extracellular matrix (ECM) exists in various biochemical and structural forms that can act either as a barrier to migrating leukocytes, in the case of basement membranes, or provide a physical scaffold supporting or guiding migration (interstitial matrix). This review focuses on basement membranes and our current knowledge of the way that leukocytes transmigrate this protein barrier, with emphasis on T lymphocytes. Recent data suggest that the classical concept of cell-matrix adhesion requires revision with respect to leukocyte-ECM interactions.

Sialoadhesin-positive macrophages bind regulatory T cells, negatively controlling their expansion and autoimmune disease progression.

An important regulatory suppressive function in autoimmune and other inflammatory processes has been ascribed to CD4(+)Foxp3(+) regulatory T cells (Tregs), which requires direct cell-cell communication between Tregs, effector T cells, and APCs. However, the molecular basis for these interactions has not yet been clarified. We show here that sialoadhesin (Sn), the prototype of the siglec family of sialic acid-binding transmembrane proteins, expressed by resident and activated tissue-infiltrating macrophages, directly binds to Tregs, negatively regulating their expansion in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE).

Endothelial basement membrane laminin alpha5 selectively inhibits T lymphocyte extravasation into the brain.

Specific inhibition of the entry of encephalitogenic T lymphocytes into the central nervous system in multiple sclerosis would provide a means of inhibiting disease without compromising innate immune responses. We show here that targeting lymphocyte interactions with endothelial basement membrane laminins provides such a possibility. In mouse experimental autoimmune encephalomyelitis, T lymphocyte extravasation correlates with sites expressing laminin alpha4 and small amounts of laminin alpha5.