InVivoMAb anti-human CD44

Catalog #BE0262
Clone:
Hermes-1
Reactivities:
Human

$164.00 - $4,280.00

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Product Details

The Hermes-1 monoclonal antibody reacts with human CD44 also known as Hermes, HCAM, and Pgp-1. CD44 is an 80-95 kDa glycoprotein that is expressed on all leukocytes, endothelial cells, hepatocytes, and mesenchymal cells. As an adhesion molecule, CD44 participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing, and hematopoiesis. CD44 is a receptor for hyaluronic acid and can also interact with other ligands, such as osteopontin, collagens, and matrix metalloproteinases (MMPs). Additionally, CD44 is involved in tumor metastasis and targeting of CD44 by antibodies has been shown to reduce the malignant activities of various neoplasms. Interestingly, high levels of the adhesion molecule CD44 on leukemic cells are essential to generate leukemia.

Specifications

Isotype Rat IgG2a, Īŗ
Recommended Isotype Control(s) InVivoMAb rat IgG2a isotype control, anti-trinitrophenol
Recommended Dilution Buffer InVivoPure pH 7.0 Dilution Buffer
Conjugation This product is unconjugated. Conjugation is available via our Antibody Conjugation Services.
Immunogen Human CD44
Reported Applications in vivo CD44 blockade in xenografts
in vitro CD44 blockade
Western blot
Immunofluorescence
Formulation PBS, pH 7.0
Contains no stabilizers or preservatives
Endotoxin <2EU/mg (<0.002EU/Ī¼g)
Determined by LAL gel clotting assay
Purity >95%
Determined by SDS-PAGE
Sterility 0.2 Ī¼M filtered
Production Purified from cell culture supernatant in an animal-free facility
Purification Protein G
RRID AB_2687741
Molecular Weight 150 kDa
Storage The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze.
in vitro CD44 blockade
Simonson, A. W., et al. (2019). "Bioresponsive peptide-polysaccharide nanogels ā€“ A versatile delivery system to augment the utility of bioactive cargo" Nanomedicine 17: 391-400. PubMed

We report the design, synthesis and efficacy of a new class of gel-like nano-carrier, or ā€˜nanogelā€™, prepared via templated electrostatic assembly of anionic hyaluronic acid (HA) polysaccharides with the cationic peptide amphiphile poly-L-lysine (PLL). Small molecules and proteins present during nanogel assembly become directly encapsulated within the carrier and are precisely released by tuning the nanogel HA:PLL ratio to control particle swelling. Remarkably, nanogels exhibit versatile and complimentary mechanisms of cargo delivery depending on the biologic context. For example, in mammalian cells, nanogels are rapidly internalized and escape the endosome to both deliver membrane-impermeable protein cargo into the cytoplasm and improve chemotherapeutic potency in drug resistant cancer cells. In bacteria, nanogels permeabilize microbial membranes to sensitize bacterial pathogens to the action of a loaded antibiotic. Thus, peptide nanogels represent a versatile, readily scalable and bio-responsive carrier capable of augmenting and enhancing the utility of a broad range of biomolecular cargoes.

in vitro CD44 blockade
Lin, C. Y., et al. (2019). "High levels of serum hyaluronan is an early predictor of dengue warning signs and perturbs vascular integrity" EBioMedicine 48: 425-441. PubMed

BACKGROUND: A main pathological feature of severe dengue virus infection is endothelial hyper-permeability. The dengue virus nonstructural protein 1 (NS1) has been implicated in the vascular leakage that characterizes severe dengue virus infection, however, the molecular mechanisms involved are not known. METHODS: A cohort of 250 dengue patients has been followed from the onset of symptoms to the recovery phase. Serum hyaluronan levels and several other clinical parameters were recorded. The effect of NS1 treatment of cultured fibroblasts and endothelial cells on the expressions of hyaluronan synthetic and catabolic enzymes and the hyaluronan receptor CD44, were determined, as have the effects on the formation of hyaluronan-rich matrices and endothelial permeability. FINDINGS: Elevated serum hyaluronan levels (ā‰„70 ng/ml) during early infection was found to be an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlated with high concentrations of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells cultured in vitro recruited CD44-expressing macrophage-like cells, suggesting a mechanism for enhancement of inflammation. In cultured endothelial cells, perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VE-cadherin and cytoskeleton re-organization, and exacerbated the NS1-induced disruption of endothelial integrity. INTERPRETATION: Pharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during moderate-to-severe dengue virus infection. FUND: This work was supported in part by grants from the Swedish Cancer Society (2018/337; 2016/445), the Swedish Research Council (2015-02757), the Ludwig Institute for Cancer Research, Uppsala University, the Ministry of Science and Technology, Taiwan (106-2314-B-037-088- and 106-2915-I-037-501-), Kaohsiung Medical University Hospital (KMUH103-3 T05) and Academy of Finland. The funders played no role in the design, interpretation or writing of the manuscript.

in vivo CD44 blockade in xenografts, in vitro CD44 blockade
Zhao, C., et al. (2019). "The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44" Oncotarget 10(61): 6561-6576. PubMed

Hyaluronan accumulation in the tumor microenvironment is associated with poor prognosis in several solid human cancers. To understand the role of stromal hyaluronan in tumor progression, we engineered 3T3HAS3, a hyaluronan-producing fibroblast cell line, by lentiviral transduction of Balb/c 3T3 cells with the human hyaluronan synthase 3 (HAS3) gene. 3T3HAS3 cells significantly enhanced tumor growth when co-grafted with MDA-MB-468 cells in nude mice. Immunohistochemical analysis of the xenograft tumors showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of MDA-MB-468 + 3T3HAS3 co-grafts was greatly reduced upon hyaluronan degradation by lentiviral transduction of a human hyaluronidase gene in 3T3HAS3 cells, or by systemic administration of pegvorhyaluronidase alfa (PEGPH20). In contrast, the growth of the co-graft tumors was not inhibited when CD44 expression was reduced or ablated by small hairpin RNA-mediated CD44 knockdown in MDA-MB-468 cells, CD44 CRISPR knockout in 3T3HAS3 cells, or by grafting these cells in CD44 knockout nude mice. Collectively, these data demonstrate that tumor growth of an engineered xenograft breast cancer model with hyaluronan-accumulating stroma can be dependent on hyaluronan and independent of CD44.

Immunofluorescence
Jokela, T., et al. (2015). "Interleukin-1beta-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes, Binding to Ezrin, and Extended, Monocyte-adhesive Hyaluronan Coats" J Biol Chem 290(19): 12379-12393. PubMed

The proinflammatory cytokine interleukin-1beta (IL-1beta) attracts leukocytes to sites of inflammation. One of the recruitment mechanisms involves the formation of extended, hyaluronan-rich pericellular coats on local fibroblasts, endothelial cells, and epithelial cells. In the present work, we studied how IL-1beta turns on the monocyte adhesion of the hyaluronan coat on human keratinocytes. IL-1beta did not influence hyaluronan synthesis or increase the amount of pericellular hyaluronan in these cells. Instead, we found that the increase in the hyaluronan-dependent monocyte binding was associated with the CD44 of the keratinocytes. Although IL-1beta caused a small increase in the total amount of CD44, a more marked impact was the decrease of CD44 phosphorylation at serine 325. At the same time, IL-1beta increased the association of CD44 with ezrin and complex formation of CD44 with itself. Treatment of keratinocyte cultures with KN93, an inhibitor of calmodulin kinase 2, known to phosphorylate Ser-325 in CD44, caused similar effects as IL-1beta (i.e. homomerization of CD44 and its association with ezrin) and resulted in increased monocyte binding to keratinocytes in a hyaluronan-dependent way. Overexpression of wild type CD44 standard form, but not a corresponding CD44 mutant mimicking the Ser-325-phosphorylated form, was able to induce monocyte binding to keratinocytes. In conclusion, treatment of human keratinocytes with IL-1beta changes the structure of their hyaluronan coat by influencing the amount, post-translational modification, and cytoskeletal association of CD44, thus enhancing monocyte retention on keratinocytes.

Western Blot, Immunofluorescence
Kim, Y. and S. Kumar. (2014). "CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility" Mol Cancer Res 12(10): 1416-1429. PubMed

The high-molecular-weight glycosaminoglycan, hyaluronic acid (HA), makes up a significant portion of the brain extracellular matrix. Glioblastoma multiforme (GBM), a highly invasive brain tumor, is associated with aberrant HA secretion, tissue stiffening, and overexpression of the HA receptor CD44. Here, transcriptomic analysis, engineered materials, and measurements of adhesion, migration, and invasion were used to investigate how HA/CD44 ligation contributes to the mechanosensing and invasive motility of GBM tumor cells, both intrinsically and in the context of Arg-Gly-Asp (RGD) peptide/integrin adhesion. Analysis of transcriptomic data from The Cancer Genome Atlas reveals upregulation of transcripts associated with HA/CD44 adhesion. CD44 suppression in culture reduces cell adhesion to HA on short time scales (0.5-hour postincubation) even if RGD is present, whereas maximal adhesion on longer time scales (3 hours) requires both CD44 and integrins. Moreover, time-lapse imaging demonstrates that cell adhesive structures formed during migration on bare HA matrices are more short lived than cellular protrusions formed on surfaces containing RGD. Interestingly, adhesion and migration speed were dependent on HA hydrogel stiffness, implying that CD44-based signaling is intrinsically mechanosensitive. Finally, CD44 expression paired with an HA-rich microenvironment maximized three-dimensional invasion, whereas CD44 suppression or abundant integrin-based adhesion limited it. These findings demonstrate that CD44 transduces HA-based stiffness cues, temporally precedes integrin-based adhesion maturation, and facilitates invasion. IMPLICATIONS: This study reveals that the CD44 receptor, which is commonly overexpressed in GBM tumors, is critical for cell adhesion, invasion, and mechanosensing of an HA-based matrix.

in vitro CD44 blockade
Qu, C., et al. (2014). "Extensive CD44-dependent hyaluronan coats on human bone marrow-derived mesenchymal stem cells produced by hyaluronan synthases HAS1, HAS2 and HAS3" Int J Biochem Cell Biol 48: 45-54. PubMed

Hyaluronan (HA), a natural extracellular matrix component, has been considered as an important constituent of the stem cell niche, and successfully used as 3D scaffolds for the chondrogenic differentiation of stem cells. However, the expression levels of HA synthases (HAS1, 2 and 3) and the synthesis of HA by stem cells have remained unknown, and were studied here in the human bone marrow-derived mesenchymal stem cells (hMSCs). Nine hMSCs from different donors were cultured as monolayers with MSC culture medium supplemented with FGF-2. The amount of HA secreted into medium was studied by an ELISA-type assay, and HA bound to cell surface by live cell microscopy. The expression of HASs was analyzed by real time RT-PCR and immunostainings. The HA receptor CD44 was studied by immunocytochemistry. An intense HA coat surrounded the plasma membrane and its protrusions in all nine hMSCs. Displacement assay with HA oligosaccharides indicated that HA coat was at least partly dependent on CD44, which showed similar, relatively high expression in all hMSCs. All HAS isoenzymes were detected, HAS1 showing the largest and HAS3 the smallest range of expression levels between the hMSCs. The secretion of HA ranged between 22.5 and 397.4 ng/10,000 cells/24h, and could not be clearly assigned to the mRNA level of a certain HAS, or a combination of the isoenzymes. This suggests that post-transcriptional and post-translational factors were involved in the adjustment of the HA secretion. In conclusion, all hMSCs expressed high levels of HAS1-3, secrete large amounts of HA, and surround themselves with a thick HA coat bound to CD44. The results suggest that hMSC has the potential for autocrine maintenance of the HA niche, important for their stemness.

Immunofluorescence
Olofsson, B., et al. (2014). "Knock-down of CD44 regulates endothelial cell differentiation via NFkappaB-mediated chemokine production" PLoS One 9(3): e90921. PubMed

A striking feature of microvascular endothelial cells is their capacity to fuse and differentiate into tubular structures when grown in three-dimensional (3D) extracellular matrices, in collagen or Matrigel, mimicking the in vivo blood vessel formation. In this study we demonstrate that human telomerase-immortalised foreskin microvascular endothelial (TIME) cells express high levels of the hyaluronan receptor CD44 and the hyaluronidase HYAL2. Knock-down of CD44 or HYAL2 resulted in an inability of TIME cells to form a tubular network, suggesting a key regulatory role of hyaluronan in controlling TIME cell tubulogenesis in 3D matrices. Knock-down of CD44 resulted in an upregulation of mRNA expression of the chemokines CXCL9 and CXCL12, as well as their receptors CXCR3 and CXCR4. This was accompanied by a defect maturation of the tubular structure network and increased phosphorylation of the inhibitor of NFkappaB kinase (IKK) complex and thus translocation of NFkappaB into the nucleus and activation of chemokine targed genes. Furthermore, the interaction between CD44 and hyaluronan determines the adhesion of breast cancer cells. In summary, our observations support the notion that the interaction between CD44 and hyaluronan regulates microvascular endothelial cell tubulogenesis by affecting the expression of cytokines and their receptors, as well as breast cancer dissemination.

in vitro CD44 blockade
Purushothaman, A. and B. P. Toole. (2014). "Serglycin proteoglycan is required for multiple myeloma cell adhesion, in vivo growth, and vascularization" J Biol Chem 289(9): 5499-5509. PubMed

Recently, it was discovered that serglycin, a hematopoietic cell proteoglycan, is the major proteoglycan expressed and constitutively secreted by multiple myeloma (MM) cells. High levels of serglycin are present in the bone marrow aspirates of at least 30% of newly diagnosed MM patients. However, its contribution to the pathophysiology of MM is unknown. Here, we show that serglycin knockdown (by approximately 85% compared with normal levels), using lentiviral shRNA, dramatically attenuated MM tumor growth in mice with severe combined immunodeficiency. Tumors formed from cells deficient in serglycin exhibited diminished levels of hepatocyte growth factor expression and impaired development of blood vessels, indicating that serglycin may affect tumor angiogenesis. Furthermore, knockdown of serglycin significantly decreased MM cell adhesion to bone marrow stromal cells and collagen I. Even though serglycin proteoglycan does not have a transmembrane domain, flow cytometry showed that serglycin is present on the MM cell surface, and attachment to the cell surface is, at least in part, dependent on its chondroitin sulfate side chains. Co-precipitation of serglycin from conditioned medium of MM cells using a CD44-Fc chimera suggests that CD44 is the cell surface-binding partner for serglycin, which therefore may serve as a major ligand for CD44 at various stages during myeloma progression. Finally, we demonstrate that serglycin mRNA expression in MM cells is up-regulated by activin, a predominant cytokine among those increased in MM patients with osteolytic lesions. These studies provide direct evidence for a critical role for serglycin in MM pathogenesis and show that targeting serglycin may provide a novel therapeutic approach for MM.

Western Blot
Picker, L. J., et al. (1989). "Monoclonal antibodies against the CD44 [In(Lu)-related p80], and Pgp-1 antigens in man recognize the Hermes class of lymphocyte homing receptors" J Immunol 142(6): 2046-2051. PubMed

An 85- to 95 kDa class of lymphocyte surface molecules, defined in man by antibodies of the Hermes series, is involved in lymphocyte binding to high endothelial venules and is likely of central importance in the process of lymphocyte homing. In this report, we have examined the relationship between these Hermes-defined ā€œhoming-receptorsā€ and two other 80 to 95 kDa lymphocyte surface molecules that have been extensively studiedā€“CD44 [In(Lu)-related p80] defined by mAb A1G3 and A3D8, and Pgp-1 defined by antibody IM7. Our findings indicate that, in man, similar or identical glycoprotein(s) are recognized by these independently and diversely obtained antibodies. All antibodies showed identical immunohistologic patterns of reactivity on a variety of lymphoid and nonlymphoid human tissues, and demonstrated similar bands on Western blots of both crude tonsil lymphocyte lysates and highly purified Hermes-1 Ag preparations. Similarly, purified CD44/p80 Ag from RBC and human serum bound Hermes-1. Preclearing of tonsil lysates with the Hermes-1 antibody removed antigenic activity for all antibodies. Cross-blocking experiments demonstrated that A3D8, IM7 (anti-Pgp-1), and Hermes-2 antibodies recognize overlapping epitopes. Finally, expression of the epitopes defined by the Hermes-1, Hermes-3, H2-7, and H3-61 antibodies on RBC was shown to be regulated by the In(Lu) gene. These findings unify several different lines of investigation, and suggest the possibility that the CD44/Pgp-1/Hermes class of molecules may serve as cell-cell or cell-substrate adhesion/recognition elements for both hematolymphoid and non-hematolymphoid cell types.