Fully closed cell sorter for immune cell therapy manufacturing.

By analyzing patients treated with adoptive immune cell therapies, various immune cell phenotypes have been found in the starting and infused materials as determinants of sustained remission. The isolation of these specific phenotypes for clinical use requires current Good Manufacturing Practice (cGMP)-compliant cell-sorting technologies with multiparameter selection capabilities. Here, we developed a cGMP-requirement-applicable fully closed cell sorter that has a suction mechanism and multiparameter detection using two laser optical settings. Negative pressure generated by a change in the chamber volume at a sorting point allows the isolation of cells of interest with high viability and purity. Our study demonstrated that this microfluidic sorter enables the isolation of cells of interest at an effective rate of 7,000 sorts per second on average. A purity of 85.5% and 77.1% effective yield with 93.7% viability was obtained when applying a target population of 35.9% in total (lymphocyte+CD8+) at 15,000 events per second (2 × 107 cells/mL). The sorted gene-modified T cells maintain largely unaltered proliferation, antigen recognition, cytokine release, and cytotoxicity functionalities.

Discovery of N-substituted 7-azaindoline derivatives as potent, orally available M1 and M4 muscarinic acetylcholine receptors selective agonists.

We designed and synthesized novel N-substituted 7-azaindoline derivatives as selective M1 and M4 muscarinic acetylcholine receptors (mAChRs) agonists. Hybridization of compound 2 with the HTS hit compound 5 followed by optimization of the N-substituents of 7-azaindoline led to identification of compound 1, which showed highly selective M1 and M4 mAChRs agonistic activity, weak human ether-a-go-go related gene inhibition, and good bioavailability in multiple animal species.

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M(1) and M(4) muscarinic acetylcholine receptors agonists.

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M1 and M4 muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M1 and M4 agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.

5-HT4 receptor stimulation leads to soluble AßPPα production through MMP-9 upregulation.

Serotonin 4 (5-HT4) receptor signaling does not only have the physiological function of improving cognition, but might also be helpful in the therapy of Alzheimer's disease (AD) through regulation of the production of soluble amyloid-ß protein precursor alpha (sAßPPα). To analyze the relationship between 5-HT4 receptor signaling and sAßPPα production, we stably transfected H4 cells with AßPP and 5-HT4 receptor (H4/AßPP/5-HT4 cells). We found that 24-h incubation with the 5-HT4 receptor agonist RS-67333 upregulates matrix metalloproteinase-9 (MMP-9). Furthermore, MMP-9 overexpression enhanced sAßPPα levels, whereas knockdown with MMP-9 siRNA decreased sAßPPα levels. When RS-67333 was injected for 10 days in Tg2576 mice, a model of amyloid-ß peptide (Aß) deposition, there was an increase in hippocampal levels of sAßPPα, C-terminal fragment α, and MMP-9, as well as a decrease in hippocampal senile plaque number and levels of the 40 amino acid peptide, Aß40. Taken all together, these experiments demonstrate that 5-HT4 receptor stimulation induces expression of MMP-9 which cleaves AßPP through α-secretase-like activity, leading to an increase of sAßPPα levels and a reduction of Aß load.

Endogenous amyloid-ß is necessary for hippocampal synaptic plasticity and memory.

OBJECTIVE: The goal of this study was to investigate the role of endogenous amyloid-ß peptide (Aß) in healthy brain. METHODS: Long-term potentiation (LTP), a type of synaptic plasticity that is thought to be associated with learning and memory, was examined through extracellular field recordings from the CA1 region of hippocampal slices, whereas behavioral techniques were used to assess contextual fear memory and reference memory. Amyloid precursor protein (APP) expression was reduced through small interfering RNA (siRNA) technique. RESULTS: We found that both antirodent Aß antibody and siRNA against murine APP reduced LTP as well as contextual fear memory and reference memory. These effects were rescued by the addition of human Aß42, suggesting that endogenously produced Aß is needed for normal LTP and memory. Furthermore, the effect of endogenous Aß on plasticity and memory was likely due to regulation of transmitter release, activation of α7-containing nicotinic acetylcholine receptors, and Aß42 production. INTERPRETATION: Endogenous Aß42 is a critical player in synaptic plasticity and memory within the normal central nervous system. This needs to be taken into consideration when designing therapies aiming at reducing Aß levels to treat Alzheimer disease.

Binding of ADAM28 to P-selectin glycoprotein ligand-1 enhances P-selectin-mediated leukocyte adhesion to endothelial cells.

ADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions.

Pericellular activation of proMMP-7 (promatrilysin-1) through interaction with CD151.

Matrix metalloproteinase-7 (MMP-7) (also known as matrilysin-1) is secreted as a proenzyme (proMMP-7) and plays a key role in the degradation of various extracellular matrix (ECM) and non-ECM molecules after activation. To identify the binding proteins related to proMMP-7 activation, a human lung cDNA library was screened by yeast two-hybrid system using proMMP-7 as bait. We identified a candidate molecule CD151, which is a member of the transmembrane 4 superfamily. Complex formation of proMMP-7 with CD151 was demonstrated by immunoprecipitation of the molecules from CaR-1 cells, a human rectal carcinoma cell line, expressing both proMMP-7 and CD151, and CD151 stable transfectants incubated with proMMP-7. Yeast two-hybrid assays using deletion mutants of proMMP-7 and CD151 suggested an interaction between the propeptide of proMMP-7 and the COOH-terminal extracellular loop of CD151. The binding activity of (125)I-labeled proMMP-7 to CD151 on the cell membranes was shown with CD151 stable transfectants. Laser-scanning confocal microscopy demonstrated that proMMP-7 colocalizes with CD151 on the cell membranes of CD151 stable transfectants and CaR-1 cells. In situ zymography using crosslinked carboxymethylated transferrin, a substrate of MMP-7, demonstrated proteinase activity on and around CD151 stable transfectants and CaR-1 cells, while the activity was abolished by their treatment with MMP inhibitors, anti-MMP-7 antibody or anti-CD151 antibody. In human lung adenocarcinoma tissues, colocalization of MMP-7 and CD151 was demonstrated on the carcinoma cells. Metalloproteinase activity was present in these tissues and could be inhibited by antibodies to MMP-7 or CD151. These data demonstrate for the first time that proMMP-7 is captured and activated on the cell membranes through interaction with CD151, and suggest the possibility that similar to the MT1-MMP/MMP-2 system, MMP-7 is involved in the pericellular activation mechanism mediated by CD151, a crucial step in proteolysis on the cell membranes under various pathophysiological conditions including cancer invasion and metastasis.

Development of in situ zymography to localize active matrix metalloproteinase-7 (matrilysin-1).

Matrix metalloproteinase-7 (MMP-7) is upregulated during carcinogenesis and its expression correlates with metastasis of human endometrial and gastrointestinal carcinomas. In the present study, we have developed a new method to localize the activity of MMP-7 within tissues. Polyethylene terephthalate films were uniformly coated with crosslinked carboxymethylated transferrin (CCm-Tf) as a substrate and incubated with frozen tissue sections mounted on the films. CCm-Tf on the films was degraded selectively by MMP-7, but showed little or no susceptibility to MMP-1, -2, -3, -9, or -13; MT1-MMP; MT3-MMP; or ADAMTS4. Although some serine proteinases such as elastase also digested CCm-Tf, CCm-Tf films impregnated with serine proteinase inhibitors prevented the digestion. When frozen sections of human endometrial carcinoma and lung carcinoma tissues were incubated on CCm-Tf films or those treated with proteinase inhibitors, the activity was detected in the carcinoma cell nests, where MMP-7 was immunolocalized. The present in situ zymography using CCm-Tf may be a useful method to analyze the functions of MMP-7 in pathophysiological conditions.

ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan.

ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including Delta Sp (Delta Arg(693)-Lys(837), lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC(50) = 110 nm), whereas no inhibition was observed with Delta Sp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC(50) = 170 nm). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.

Matrix metalloproteinases cleave connective tissue growth factor and reactivate angiogenic activity of vascular endothelial growth factor 165.

Vascular endothelial growth factor (VEGF), a potent angiogenic mitogen, plays a crucial role in angiogenesis under various pathophysiological conditions. We have recently demonstrated that VEGF(165), one of the VEGF isoforms, binds connective tissue growth factor (CTGF) and that its angiogenic activity is inhibited in the VEGF(165).CTGF complex form (Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H., Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K. and Okada, Y. (2002) FASEB J. 16, 219-221). In the present study, we further examined the susceptibility of the VEGF(165).CTGF complex to matrix metalloproteinases (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4 (aggrecanase-1), and serine proteinases, and evaluated the recovery of the angiogenic activity of VEGF(165) after the treatment. Among the MMPs, MMP-1, -3, -7, and -13 processed CTGF of the complex into the major NH(2)- and COOH-terminal fragments, whereas VEGF(165) was completely resistant to the MMPs. On the other hand, elastase and plasmin cleaved both CTGF and VEGF(165) of the complex, but they were completely resistant to ADAMTS4. By digestion of the immobilized VEGF(165).CTGF complex with MMP-3 or MMP-7, both NH(2)- and COOH-terminal fragments of CTGF were dissociated and released from the complex into the liquid phase. The in vitro angiogenic activity of VEGF(165) blocked in the VEGF(165).CTGF complex was reactivated to original levels after CTGF digestion of the complex with MMP-1, -3, and -13. Recovery of angiogenic activity was further confirmed by in vivo angiogenesis assay using a Matrigel injection model in mice. These results demonstrate for the first time that CTGF is a substrate of MMPs and that the angiogenic activity of VEGF(165) suppressed by the complex formation with CTGF is recovered through the selective degradation of CTGF by MMPs. MMPs may play a novel role through CTGF degradation in VEGF-induced angiogenesis during embryonic development, tissue maintenance, and/or pathological processes of various diseases.

Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis.

Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two-hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity-binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 +/- 11 nM and 125 +/- 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type-1 repeat (TSP-1) domain of CTGF binds to the exon 7-coded region of VEGF165 and that the COOH-terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full-length CTGF and the deletion mutant possessing the TSP-1 domain inhibited VEGF165-induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein-to-protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.