Immunogenicity of a multi-epitope DNA vaccine against hantavirus

Hemorrhagic fever with renal syndrome (HFRS) is a severe epidemic disease caused by hantaviruses including Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus. Three of the four HFRS hantaviruses, HTNV, SEOV, and PUUV are found in China. Currently, there is no effective strategy available to reduce infection risk. In this study, we constructed a multi-epitope chimeric DNA vaccine that encodes expressing 25 glycoprotein epitopes from SEOV, HTNV and PUUV (designated as SHP chimeric gene). Vaccination of BALb/c mice with SHP multi-epitope chimeric DNA vaccine led to a dramatic augmentation of humoral and cellular responses. The SHP vaccine DNA was detected in many organs but not for more than 60 d. There was no risk of mutation due to integration. Thus, the SHP multi-epitope chimeric DNA vaccine is a potential effective and safe DNA vaccine against infection by SEOV, HTNV, and PUUV.     

Co‐circulation of three pathogenic hantaviruses: Puumala,Dobrava, and Saaremaa in Hungary

Hantaviruses (Bunyaviridae) cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus (cardio)pulmonary syndrome (HCPS) in the Americas. HFRS is caused by Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), Saaremaa virus (SAAV), and Puumala virus (PUUV). Of those, only HTNV is not present in Europe. In recent years, hantaviruses, described in other parts of Europe, were also detected at various locations in Hungary. To study the genetic properties of Hungarian hantaviruses in detail, sequences of the viral S and M segments were recovered from bank voles (Myodes glareolus), yellow‐necked mice (Apodemus flavicollis), and striped field mice (Apodemus agrarius) trapped in the Transdanubian region. As expected, the sequences recovered belonged, respectively, to PUUV (two strains), DOBV (one strain), and SAAV (one strain). On phylogenetic trees two new Hungarian PUUV strains located within the well‐ supported Alpe‐Adrian (ALAD) genetic lineage that included also Austrian, Slovenian, and Croatian strains. Analysis of the Hungarian SAAV and DOBV genetic variants showed host‐specific clustering and also geographical clustering within each of these hantavirus species. Hungarian SAAV and DOBV strains were related most closely to strains from Slovenia (Prekmurje region). This study confirms that multiple hantaviruses can co‐circulate in the same locality and can be maintained side‐by‐side in different rodent species. J. Med. Virol. 81:2045–2052, 2009. © 2009 Wiley‐Liss, Inc.     

A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection

Hantaan virus (HTNV) is a pathogenic hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). HTNV infection is mediated by αvβ3 integrin. We used protein blots of Vero E6 cell homogenates to demonstrate that radiolabeled HTNV virions bind to gC1qR/p32, the acidic 32-kDa protein known as the receptor for the globular head domain of complement C1q. RNAi-mediated suppression of gC1qR/p32 markedly reduced HTNV binding and infection in human lung epithelial A549 cells. Conversely, transient expression of either simian or human gC1qR/p32 rendered non-permissive CHO cells susceptible to HTNV infection. These results suggest an important role for gC1qR/p32 in HTNV infection and pathogenesis.     

Pathogenic hantaviruses selectively inhibit β<Subscript>3</Subscript> integrin directed endothelial cell migration

Summary.  Hantaviruses cause two diseases of man, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Pathogenic and non-pathogenic hantaviruses use β₃ and β₁ integrins, respectively, to enter endothelial cells. β₃ integrins were recently reported to bind receptors that regulate vascular permeability suggesting that hantavirus β₃ integrin interactions may regulate endothelial cell function and contribute to viral pathogenesis. In this study we investigated the ability of pathogenic and non-pathogenic hantaviruses to regulate β₃ and β₁ integrin directed endothelial cell functions. We found that pathogenic NY-1, SNV, HTN, SEO and PUU viruses blocked endothelial cell migration on β₃, but not β₁, integrin ligands. Migration is similarly inhibited by antibodies to β₃ integrins which selectively block vitronectin directed endothelial cell migration. As a result, the ability of endothelial cells to migrate on integrin ligands was selectively inhibited by only pathogenic hantaviruses. Infection by NY-1 virus inhibited endothelial cell migration as early as 24–48 h post-infection. In contrast, non-pathogenic PH and TUL viruses had no effect on the ability of endothelial cells to migrate on either β₃ or β₁ integrin ligands from 1 to 5 days post-infection. These findings indicate that only hantaviruses which use β₃ integrins, and are associated with HPS and HFRS diseases, functionally dysregulate endothelial cell migration. These findings further demonstrate that hantaviruses regulate only β₃ integrin directed endothelial cell functions and have no effect on β₁ integrin functions. Since β₃ integrins are linked to changes in vascular permeability and the maintenance of vascular integrity, these findings suggest a means by which hantavirus usage and regulation of β₃ integrins may contribute to hantavirus pathogenesis. Received January 23, 2002; accepted May 20, 2002     

Chronic encephalomyelitis virus exhibits cellular tropism and evades pDCs by binding to sialylated integrins as the cell surface receptors

Theiler's murine encephalomyelitis virus (TMEV) causes a chronic demyelinating disease similar to multiple sclerosis in mice. Although sialic acids have been shown to be essential for TMEV attachment to the host, the surface receptor has not been identified. While type I interferons play a pivotal role in the elimination of the chronic infectious Daniel (DA) strain, the role of plasmacytoid dendritic cells (pDCs) is controversial. We herein found that TMEV binds to conventional DCs but not to pDCs. A glycomics analysis showed that the sialylated N-glycan fractions were lower in pDCs than in conventional DCs, indicating that pDCs are not susceptible to TMEV infection due to the low levels of sialic acid. TMEV capsid proteins contain an integrin recognition motif, and dot blot assays showed that the integrin proteins bind to TMEV and that the viral binding was reduced in the desialylated α_Xβ₂. α_Xβ₂ protein suppressed TMEV replication in vivo, and TMEV co-localized with integrin α_M at the cell membrane and TLR 3 in the cytoplasm, suggesting that α_M serves as the viral attachment and entry. These results show that the chronic encephalomyelitis virus utilizes sialylated integrins as cell surface receptors, leading to cellular tropism to evade pDC activation.     

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9–10) affects its integrin specificity. Through production of FN III9–10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α₅β₁ and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α₅β₁ integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α₅β₁ has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α₅β₁ versus α_vβ₃ results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.     

α6 integrins participate in pro-T cell homing to the thymus

During embryogenesis, colonization of the thymic rudiment by hemopoietic progenitor cells depends on the adhesion of these cells to the jugular endothelium. Previously, we showed that progenitor T cells (pro-T cells) interact with α₆ integrins present on vascular endothelium. Here, we demonstrate that anti-α₆ integrin antibodies reduced the number of thymocytes up to 80 % in a congenic mouse model for thymus colonization by pro-T cells. In organotypic thymus cultures, the anti-α₆ integrin antibodies did not influence T cell development and proliferation. From this, we conclude that α₆ integrin participates in thymus homing. During mouse thymus ontogeny, α₆ integrin mRNA and protein expression was found as early as day 10 of development; at day 11, perithymic endothelial cells were α₆ integrin positive. Two α₆ integrin mRNA exist which are produced by alternative exon usage. The longer form, α₆, integrin, predominates during early embryonic stages, while the shorter α_6A form was present later during development. Although α₆, integrins can be displayed by immature thymocytes, strongest expression was found on intra- and perithymic vascular endothelium. These data suggest that α₆ integrins are involved in the homing of pro-T cells to the developing thymus by mediating adhesion of pro-T cells to the vascular endothelium.     

Distinct structural and functional epitopes of the αEβ7 integrin

Intestinal intraepithelial lymphocytes (iIEL) are predominantly CD3⁺, CD8⁺ T lymphocytes located above or adjacent to the mucosal basement membrane. Although they are positioned to interact with intercellular luminal antigen or with enterocytes, the function of iIEL remains unknown. Most (> 85%) of the iIEL express the α^Eβ₇ integrin which appears to be involved in the adhesion of lymphocytes to epithelial cells. We report the characterization of three monoclonal antibodies (mAb) termed αE7-1, αE7-2, and αE7-3, that react with the α^Eβ₇ integrin recognized by the previously described mAb HML-1 as demonstrated by identical sodium dodecyl sulfate – polyacrylamide gel electrophoresis mobility and charge. Flow cytometric analysis of antibody cross-blocking indicated that these mAb recognize distinct epitopes of α^Eβ₇. While all of the mAb were capable of blocking the adhesion of cultured iIEL to a breast epithelial cell line, only HML-1 and αE7-1 (which recognize an identical or closely related epitope) were co-stimulatory with suboptimal concentrations of anti-CD3 mAb in inducing proliferation of cultured iIEL. Thus, these mAb appear to recognize functionally distinct epitopes of α^Eβ₇ and will be useful to study relationships between the structure and function of this integrin.     

Hantavirus infections in Europe and their impact on public health

Hantaviruses (genus Hantavirus, family Bunyaviridae) are enveloped tri‐segmented negative‐stranded RNA viruses each carried by a specific rodent or insectivore host species. Several different hantaviruses known to infect humans circulate in Europe. The most common is Puumala (PUUV) carried by the bank vole; another two important, genetically closely related ones are Dobrava–Belgrade (DOBV) and Saaremaa viruses (SAAV) carried by Apodemus mice (species names follow the International Committee on Taxonomy of Viruses nomenclature). Of the two hantaviral diseases, hemorrhagic fever with renal syndrome (HFRS) and hantaviral cardiopulmonary syndrome, the European viruses cause only HFRS: DOBV with often severe symptoms and a high case fatality rate, and PUUV and SAAV more often mild disease. More than 10,000 HFRS cases are diagnosed annually in Europe and in increasing numbers. Whether this is because of increasing recognition by the medical community or due to environmental factors such as climate change, or both, is not known. Nevertheless, in large areas of Europe, the population has a considerable seroprevalence but only relatively few HFRS cases are reported. Moreover, no epidemiological data are available from many countries. We know now that cardiac, pulmonary, ocular and hormonal disorders are, besides renal changes, common during the acute stage of PUUV and DOBV infection. About 5% of hospitalized PUUV and 16%–48% of DOBV patients require dialysis and some prolonged intensive‐care treatment. Although PUUV–HFRS has a low case fatality rate, complications and long‐term hormonal, renal, and cardiovascular consequences commonly occur. No vaccine or specific therapy is in general use in Europe. We conclude that hantaviruses have a significant impact on public health in Europe. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of integrin,cadherin, and catenin expression in squamous cell carcinomas of the oral cavity

In addition to their role in maintenance of tissue integrity, cell adhesion molecules regulate the growth and differentiation of stratified squamous epithelia. Reduced expression of E-cadherin and the α₂β₁, α₃β₁ and α₆β₄ integrins is already reported to correlate with poor histological differentiation in oral squamous cell carcinomas. However, it is not clear how closely cadherin and integrin loss are related in any given tumour, nor whether cadherin loss is correlated with changes in expression of the cytoplasmic regulatory proteins known as catenins. Double-label immunofluorescence has been used to stain a panel of 22 oral squamous cell carcinomas with antibodies to ten proteins, including E- and P-cadherin, the major keratinocyte integrin subunits, and α-, β- and γ-catenin. Overall, E-cadherin expression and integrin expression correlated well with tumour grade, while P-cadherin staining was more variable. All tumours, regardless of differentiation status, showed reduced staining for at least two of the catenins, implying that the adhesive function of E- and P-cadherin could be impaired even when cadherin expression is normal. It is concluded that in all squamous cell carcinomas, regardless of degree of histological differentiation, there is some perturbed expression of cell adhesion molecules and that integrin and E-cadherin loss are closely related. © 1998 John Wiley & Sons, Ltd.     

Down-regulation of MFG-E8 by RNA interference combined with doxorubicin triggers melanoma destruction

The pathogenic mechanism of malignant melanoma involves the dynamic interplay of transformed cell and normal host cell, but cancer treatments always target each partition separately. In the tumor microenvironment, milk fat globule epidermal growth factor-8 (MFG-E8) is a secreted glycoprotein highly expressed in the vertical growth phase of melanoma, leading to tumor progression through coordinated α_vβ₃ and α_vβ₅ integrin signaling in tumor cells and host cells. Doxorubicin (Dox) is one of the most widely used antitumor drugs against a lot of solid tumors, including melanoma. In this work, Dox was used to combine with down-regulation of MFG-E8 by RNA interference (RNAi) in order to determine the synergistic effect of the antitumor activity in vivo. And the possible mechanisms were investigated. Results showed that combination group (MFG-E8 RNAi plus Dox) could inhibit the growth of melanoma more effectively than monotherapy or control groups. We found that the combination treatment induced more tumor cell apoptosis and inhibited more neovascularization than other groups. Moreover, this combination treatment attenuated CD4⁺ CD25⁺ Foxp3⁺ Treg cells in tumor-infiltrating lymphocytes compared with other groups. Our findings suggested that MFG-E8 down-regulation enhanced the antitumor function of chemotherapy through coordinated cell apoptosis and immune-mediated mechanisms, which might be a feasible way for cancer therapy.     

Dysregulation of the β3 integrin-VEGFR2 complex in Hantaan virus–directed hyperpermeability upon treatment with VEGF

Hantaviruses infect human endothelial cells (ECs) and are known to cause vascular-permeability-based diseases, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The αvβ3 integrins, which are highly expressed on the surface of ECs, serve as hantavirus receptors. Specifically, the β3 integrin and vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) form a functional complex and interact with each other. Signaling through this complex causes cytoskeletal reorganization, which is one of the most important mechanisms underlying hyperpermeability. In this study, we show that VEGF dramatically enhances Hantaan virus (HTNV)-directed permeability and increases the reorganization of the cytoskeleton and the disruption of junctional organizations in an EC monolayer at 3 days postinfection. HTNV infection reduced the effect of VEGF on adhesion, migration, and the upregulation of β3 expression, but the infection alone upregulated the expression of β3 and VEGFR2. These results indicate that in addition to its role in blocking β3 integrin activation as reported previously, HTNV blocks the function of the complex of VEGFR2 and β3 integrin, and the dysfunction of the complex may contribute to cytoskeletal reorganization in an HTNV-directed hyperpermeability response to VEGF.     

Antiviral effect of human saliva against hantavirus

Hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome are zoonotic diseases caused by rodent borne hantaviruses. Transmission to humans occurs usually by inhalation of aerozolized virus‐contaminated rodent excreta. Although human‐to‐human transmission of Andes hantavirus has been observed, the mode of transmission is currently not known. Saliva from Puumala hantavirus (PUUV)‐infected patients was shown recently to contain viral RNA. To test if human saliva interferes with hantavirus replication, the effect of saliva and salivary proteins on hantavirus replication was studied. It was observed that saliva from healthy individuals reduced Hantaan hantavirus (HTNV) infectivity, although not completely. Furthermore, HTNV was resistant against the antiviral capacity of histatin 5, lysozyme, lactoferrin, and SLPI, but was inhibited by mucin. Inoculation of bank voles (Myodes glareolus) with HFRS‐patient saliva, positive for PUUV‐RNA, did not induce sero‐conversion. In conclusion, no evidence of infectious virus in patient saliva was found. However, the in vitro experiments showed that HTNV, the prototype hantavirus, is insensitive to several antiviral salivary proteins, and is partly resistant to the antiviral effect of saliva. It therefore remains to be shown if human saliva might contain infectious virions early during infection, that is, before seroconversion. J. Med. Virol. 80:2122–2126, 2008. © 2008 Wiley‐Liss, Inc.     

B cell maintenance of subcapsular sinus macrophages protects against a fatal viral infection independent of adaptive immunity

Neutralizing antibodies have been thought to be required for protection against acutely cytopathic viruses, such as the neurotropic vesicular stomatitis virus (VSV). Utilizing mice that possess B cells but lack antibodies, we show here that survival upon subcutaneous (s.c.) VSV challenge was independent of neutralizing antibody production or cell-mediated adaptive immunity. However, B cells were absolutely required to provide lymphotoxin (LT) α1β2, which maintained a protective subcapsular sinus (SCS) macrophage phenotype within virus draining lymph nodes (LNs). Macrophages within the SCS of B cell-deficient LNs, or of mice that lack LTα1β2 selectively in B cells, displayed an aberrant phenotype, failed to replicate VSV, and therefore did not produce type I interferons, which were required to prevent fatal VSV invasion of intranodal nerves. Thus, although B cells are essential for survival during VSV infection, their contribution involves the provision of innate differentiation and maintenance signals to macrophages, rather than adaptive immune mechanisms.     

Detection of VSV(MuLV) pseudotypes by an immunobiochemical technique

Vesicular stomatitis virus (murine leukemia virus) (VSV(MuLV)) pseudotypes containing a [³H]uridine-labeled VSV RNA genome and MuLV envelope glycoproteins (gp70) were produced by phenotypic mixing of the two viruses. In order to better detect such pseudotypes, an immunobiochemical (IB) technique was developed. [³H]Uridine-labeled virus progeny of the dual virus infection was immunoprecipitated by monospecific MuLV gp70 antibodies complexed with fixed Staphylococcus aureus. The immunoprecipitated ³H-labeled genomic RNA was identified as that of VSV by its sedimentation coefficient, by the lack of polyadenylate, and by molecular hybridization with complementary VSV RNA. By the IB technique, approximately 11% of the progeny of the dual virus infection were found to be VSV(MuLV). By neutralization and other biological assays, however, only 0.1% of the progeny were found to be VSV(MuLV) pseudotypes. Apparently, the IB technique is capable of detecting VSV pseudotypes encapsidated with only a few molecules of MuLV gp70. The IB technique, therefore, offers a quantitative and molecular technique for the detection of VSV(MuLV) pseudotypes and can be modified to detect other viral pseudotypes when other assays are lacking. In spite of its sensitivity however, the IB technique did not detect the formation of MuLV (VSV) pseudovirions among the virus progeny of the dual virus infection. These results confirmed a similar observation made previously using immunoelectron microscopy.     

Expression of integrin subunits in the human infant breast correlates with morphogenesis and differentiation

Integrins are widely expressed on normal tissues and their function is considered critical directly or indirectly with the control of cell growth and differentiation. Also, they are likely to play a crucial role in cell–matrix interactions during development. As the human breast develops after birth, it provides a rare opportunity in which to study human organogenesis. We have examined the distribution of integrins in the human infant breast with the aim of elucidating the possible role of these molecules in morphogenesis and differentiation. Necropsy breast specimens from six male and eight female infants, ranging in age from 1 day to 9 months, were used in this study. Cryostat sections were stained by the avidin-biotin complex technique, using a panel of monoclonal antibodies (MAbs) which recognize β₁, α₂, α₆, β₄, α_v, and α_vβ₃ integrin chains, which are candidate molecules for a role in mammory morphogenesis. MAbs to β₁ (DH12) and α₂ (HAS3) showed positive membrane and cytoplasmic staining of basal cells and luminal epithelial cells. In addition, positive staining for the β₁ integrin chain was found on fibroblasts. A MAb which recognizes the α₆ chain (MP4F10) showed positive staining of the basal cells and heterogeneous staining of the luminal epithelial cells, whilst β₄ chain (439-9B) showed positive staining in the basement membrane domain of the basal cells with no staining of the luminal epithelial cells. There was a positive correlation between the intensity of expression and the structural development of the ductal system, with integrin expression reduced or absent in the end buds and lateral buds. These data provide evidence that some integrin molecules are expressed in a pattern that correlates with the morphological and functional differentiation of the normal mammary gland. Changes in the expression and function of integrins may have an inductive role in the development of the normal mammary gland.     

Use of Vesicular Stomatitis Virus Pseudotypes Bearing Hantaan or Seoul Virus Envelope Proteins in a Rapid and Safe Neutralization Test

A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVΔG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVΔG*G. Pseudotype VSV with the Hantaan (VSVΔG*-HTN) or Seoul (VSVΔG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10⁵ to 10⁶/ml. The infectivity of VSVΔG*-HTN and VSVΔG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVΔG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVΔG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4°C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.     

Immunodetection of Cell Adhesion Molecules in Rat Sertoli Cell Cultures

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell-matrix and cell-to-cell interactions in Sertoli cell cultures was examined. METHOD OF STUDY: Immunohistochemical and Western blot techniques were applied to rat Sertoli cell cultures using specific antibodies to α₃, α₅, and α₆ integrin subunits; NCAM; and Cadherins. RESULTS: Expression of α₃ and α₆ integrin subunits (mainly laminin receptors) and lack of expression of α₅ integrin subunit (fibronectin receptor) was observed in Sertoli cells by immunohistochemistry. These cells also expressed neural CAM (NCAM) and N-cadherin. By Western blot analysis, Sertoli cell extracts reacted with antibodies to α₃ integrin subunit revealed a band approximately 130 kDa, whereas no expression of α5 integrin subunit was detected. Cell extracts incubated with antibodies to pan Cadherin exhibited a band approximately 120 kDa, whereas bands of 180, 140, and 120 kDa were observed with antibodies to NCAM. CONCLUSION: New data about the expression of receptors for extracellular matrix proteins (α₃ and α₆ integrin subunits) as well as cell-to-cell adhesion molecules (NCAM and Cadherins) are reported in rat Sertoli cell cultures.     

Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin αM290β7 (αEβ7)

The integrin α_M290β7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn²⁺ strongly promoted adhesion, Ca²⁺ was ineffective and Mg²⁺ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by α_M290β7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-γ and tumor necrosis factor-α to induce ICAM-1 expression and, in addition, α_M290β7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor β. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the α_M290β7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for α_M290β7.     

Characterization of valvular interstitial cell function in three dimensional matrix metalloproteinase degradable PEG hydrogels

Valvular interstitial cells (VICs) maintain functional heart valve structure and display transient fibroblast and myofibroblast properties. Most cell characterization studies have been performed on plastic dishes; while insightful, these systems are limited. Thus, a matrix metalloproteinase (MMP) degradable poly(ethylene glycol) (PEG) hydrogel system is proposed in this communication as a useful tool for characterizing VIC function in 3D. When encapsulated, VICs attained spread morphology, and proliferated and migrated as shown through real-time cell microscopy. Additionally, fibronectin derived pendant RGD was incorporated into the system to promote integrin binding. As RGD concentration increased from 0 to 2000 μm, VIC process extension and integrin α_vβ₃ binding increased within two days. By day 10, integrin binding was equalized between conditions. VIC morphology and rate of process extension were also increased through decreasing the hydrogel matrix density presented to the cells. VIC differentiation in response to exogenously delivered transforming growth factor-beta1 (TGF-β1) was also examined within the hydrogel networks. TGF-β1 increased expression of alpha smooth muscle actin (αSMA) and collagen-1 at both the mRNA and protein level by day 2 of culture, indicating myofibroblast differentiation, and was sustained over the course of the study (2 weeks). These studies demonstrate the utility, flexibility, and biological activity of this MMP-degradable system for the characterization of VICs, an important cell population for tissue engineering viable valve replacements and understanding valvular pathobiology.