Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection

The respiratory syncytial virus (RSV) causes potentially fatal lower respiratory tract infection in infants. The molecular mechanism of RSV infection is unknown. Our data show that RSV colocalizes with intercellular adhesion molecule-1 (ICAM-1) on the HEp-2 epithelial cell surface. Furthermore, a neutralizing anti-ICAM-1 mAb significantly inhibits RSV infection and infection-induced secretion of proinflammatory chemokine RANTES and mediator ET-1 in HEp-2 cells. Similar decrease in RSV infection is also observed in A549, a type-2 alveolar epithelial cell line, and NHBE, the normal human bronchial epithelial cell line when pretreated with anti-ICAM-1 mAb prior to RSV infection. Incubation of virus with soluble ICAM-1 also significantly decreases RSV infection of epithelial cells. Binding studies using ELISA indicate that RSV binds to ICAM-1, which can be inhibited by an antibody to the fusion F protein and also the recombinant F protein can bind to soluble ICAM-1, suggesting that RSV interaction with ICAM-1 involves the F protein. It is thus concluded that ICAM-1 facilitates RSV entry and infection of human epithelial cells by binding to its F protein, which is important to viral replication and infection and may lend itself as a therapeutic target.     

Immunogenicity of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein), produced in Spodoptera frugiperda (Sf9) cells following infection with a baculovirus vector containing the full-length (1.6 kb) glycoprotein gene, provided very limited protection in rainbow trout Oncorhynchus mykiss challenged with IHNV. Fish were injected intraperitoneally (i.p.) with Sf9 cells grown at 20 degrees C (RecGlow) or 27 degrees C (RecGhigh) expressing the glycoprotein gene. Various antigen (Ag) preparations were administered to adult rainbow trout or rainbow trout fry. Sera collected from adult fish were evaluated for IHNV neutralization activity by a complement-dependent neutralization assay. Anti-IHNV neutralizing activity was observed in sera, but the percent of fish responding was significantly lower (p < 0.05) in comparison to fish immunized with a low virulence strain of IHNV (LV-IHNV). A small number of fish immunized with RecGlow or RecGhigh possessed IHNV G protein specific antibodies (Abs) in their serum. Cumulative mortality (CM) of rainbow trout fry (mean weight, 1 g) vaccinated by i.p. injection of freeze/thawed Sf9 cells producing RecGlow was 18% in initial trials following IHNV challenge. This level of protection was significant (p < 0.05) but was not long lasting, and neutralizing Abs were not detected in pooled serum samples. When trout fry (mean weight, 0.6 g) were vaccinated with supernatant collected from sonicated Sf9 cells, Sf9 cells producing RecGlow, or Sf9 cells producing RecGhigh, CM averaged 46%. Protection was enhanced over negative controls, but not the positive controls (2% CM), suggesting that in the first trial soluble cellular proteins may have provided some level of non-specific protection, regardless of recombinant protein expression. Although some immunity was elicited in fish, and RecGlow provided short-term protection from IHNV, Ab-mediated protection could not be demonstrated. The results suggest that recombinant G proteins produced in insect cells lack the immunogenicity associated with vaccination of fish with an attenuated strain of IHNV.     

Fibronectin binding protein A of Staphylococcus aureus can mediate human T lymphocyte adhesion and coactivation

The extracellular matrix protein fibronectin (FN) mediates the adhesion of bacteria as well as T lymphocytes. Mammalian cells express integrins alpha(4)beta(1) and alpha(5)beta(1) as the major FN-binding cell surface receptors. Bacteria such as Staphylococcus aureus, also express FN-binding receptors that are important for adherence to host tissue and initiation of infection. The S. aureus FN-binding protein, FnbpA, has been previously identified, and recombinant proteins that correspond to distinct functional regions of this protein have been made. Three recombinant truncated forms of FnbpA, rFnbpA(37-881), rFnbpA(37-605), and rFnbpA(620-881), were examined for effects on in vitro adhesion and coactivation of human T lymphocytes. These proteins, when coimmobilized with anti-CD3 mAb, activated T lymphocyte proliferation. The coactivation signal generated by the rFnbpA proteins required medium containing serum with FN. Furthermore, the costimulatory signal could be restored in FN-depleted serum when the rFnbpAs were preloaded with soluble FN. Monoclonal Ab blocking studies revealed that integrin alpha(5)beta(1) is the major receptor responsible for the rFnbpA costimulatory signal. Shear flow cell detachment assays confirmed that lymphocytes can bind to FN captured by the rFnbpA proteins. These results suggest that the S. aureus rFnbpA can interact with integrin alpha(5)beta(1) via an FN bridge to mediate adhesion and costimulatory signals to T lymphocytes.     

Fibronectin enhances macrophage association with invasive forms of Trypanosoma cruzi

Treatment of either mouse peritoneal macrophages (MPH) or invasive blood forms of Trypanosoma cruzi with human plasma fibronectin (FN) significantly enhanced their association (a term to mean surface attachment and parasite internalization) with the untreated counterpart in a dose-dependent manner. This effect involved increases in the percentage of MPH that associated with the parasites and in the number of parasites per MPH. By using indirect immunofluorescence, the percentages of FN-positive MPH and FN-positive parasites found in preparations of these cells were 26 and 13%, respectively, and increased to 70 and 73%, respectively, after incubation with FN for 60 min and multiple washings. These results demonstrated the presence of FN itself and FN-binding sites on the surface of MPH and T. cruzi. Incubation of FN-treated MPH and FN-treated parasites with gelatin, for which FN has a binding site, significantly reduced the stimulatory effect of FN. A reduction was also seen when FN-treated MPH were incubated with anti-FN antibody before adding the parasites. These observations suggested that FN might enhance association by bridging the interacting cells. The presence of excess soluble FN during MPH-parasite interaction also inhibited the association, possibly by blocking FN receptors on the MPH and parasite surfaces. Pretreatment of the MPH with FN enhanced the capacity of these cells to associate with either untreated latex beads or killed T. cruzi. These findings indicated, on the one hand, that the FN-mediated enhancement was not unique to living T. cruzi and, on the other, that this enhancement was not likely due to an FN-induced alteration of the MPH membrane that would render it more susceptible to active penetration by the parasites. Taken together, these results suggest that FN, produced by MPH, may play a role in infection of this cell type by T. cruzi.     

Essential role of the NV protein of Novirhabdovirus for pathogenicity in rainbow trout

Novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) are fish rhabdoviruses that, in comparison to the other rhabdoviruses, contain an additional gene coding for a small nonvirion (NV) protein of unassigned function. A recombinant IHNV with the NV gene deleted but expressing the green fluorescent protein (rIHNV-Delta NV) has previously been shown to be efficiently recovered by reverse genetics (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000). However, preliminary experiments suggested that the growth in cell culture of rIHNV-Delta NV was affected by the NV deletion. In the present study, we show that the growth in cell culture of rIHNV-Delta NV is indeed severely impaired but that a normal growth of rIHNV-Delta NV can be restored when NV is provided in trans by using fish cell clones constitutively expressing the NV protein. These results indicate that NV is a protein that has a crucial biological role for optimal replication of IHNV in cell culture. Although IHNV and VHSV NV proteins do not share any significant identity, we show here that both NV proteins play a similar role since a recombinant IHNV virus, rIHNV-NV(VHSV), in which the IHNV NV open reading frame has been replaced by that of VHSV, was shown to replicate as well as the wild-type (wt) IHNV into fish cells. Finally, data provided by experimental fish infections with the various recombinant viruses strongly suggest an essential role of the NV protein for the pathogenicity of IHNV. Furthermore, we show that juvenile trout immunized with NV-knockout IHNV were protected against challenge with wt IHNV. That opens a new perspective for the development of IHNV attenuated live vaccines.     

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.     

alpha-Mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE). A possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells

Incubation of a Spodoptera frugiperda (IPLB-SF-21AE) cell extract with the oligosaccharide Man9GlcNAc2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant Asn residue in the nascent protein, results in its trimming to Man6GlcNAc2, an intermediate that is relatively stable to further alpha-D-mannosidase action in these cells. On the other hand, incubation of a similar extract from cells that had been infected for various times with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus) or a recombinant baculovirus (r-BAC)/human plasminogen (HPg) construct employed for expression of HPg led to rapid trimming of Man6GlcNAc2 to Man5GlcNAc2 and Man3GlcNAc2. These latter reactions displayed temporal effects, in that an enhancement of this latter trimming process occurred as a function of the time of infection of the cells with the wild-type and recombinant viral constructs. We have previously demonstrated that the nature of the oligosaccharide assembled on Asn289 of HPg expressed in several lepidopteran insect cell lines was dependent on the time of infection of the cells with r-BAC/HPg and that the amount of complex glycan found on this recombinant protein increased with an increase in infection times [Davidson, D. J., & Castellino, F. J. (1991) Biochemistry 30, 6167-6174].(ABSTRACT TRUNCATED AT 250 WORDS)     

iso-DGR Sequences Do Not Mediate Binding of Fibronectin N-terminal Modules to Adherent Fibronectin-null Fibroblasts

Fibronectin (FN) without an RGD sequence (FN-RGE), and thus lacking the principal binding site for α5β1 integrin, is deposited into the extracellular matrix of mouse embryos. Spontaneous conversion of ²⁶³NGR and/or ⁵⁰¹NGR to iso-DGR possibly explains this enigma, i.e. ligation of iso-DGR by αvβ3 integrin may allow cells to assemble FN. Partial modification of ²⁶³NGR to DGR or iso-DGR was detected in purified plasma FN by mass spectrometry. To test functions of the conversion, one or both NGR sequences were mutated to QGR in recombinant N-terminal 70-kDa construct of FN (70K), full-length FN, or FN-RGE. The mutations did not affect the binding of soluble 70K to already adherent fibroblasts or the ability of soluble 70K to compete with non-mutant FN or FN-RGE for binding to FN assembly sites. Non-mutant FN and FN-N263Q/N501Q with both NGRs mutated to QGRs were assembled equally well by adherent fibroblasts. FN-RGE and FN-RGE-N263Q/N501Q were also assembled equally well. Although substrate-bound 70K mediated cell adhesion in the presence of 1 mm Mn²⁺ by a mechanism that was inhibited by cyclic RGD peptide, the peptide did not inhibit 70K binding to cell surface. Mutations of the NGR sequences had no effect on Mn²⁺-enhanced cell adhesion to adsorbed 70K but caused a decrease in cell adhesion to reduced and alkylated 70K. These results demonstrate that iso-DGR sequences spontaneously converted from NGR are cryptic and do not mediate the interaction of the 70K region of FN with the cell surface during FN assembly.     

Induction of chemokine secretion and enhancement of contact-dependent macrophage cytotoxicity by engineered expression of granulocyte-macrophage colony-stimulating factor in human colon cancer cells

We investigated the role of tumor cell-derived GM-CSF in recruitment and tumoricidal activation of tissue macrophages. Transfection of the murine GM-CSF gene into KM12SM human colon cancer cells decreased the tumorigenicity of transfected cells and nontransfected bystander colon cancer cells in nude mice. Sequential tissue sections from sites injected with high GM-CSF-producing tumor cells (but not from nontransfected or low GM-CSF-producing cells) demonstrated a dense infiltration of polymorphonuclear cells (PMN), followed by infiltration of macrophages, which correlated with expression of the macrophage-inflammatory protein-1alpha and the monocyte chemoattractant protein-1 (MCP-1) in mouse PMN and macrophages. GM-CSF-producing KM12SM cells were highly sensitive to lysis by mouse macrophages and also increased macrophage-mediated lysis of bystander nontransfected KM12SM cells. The incubation of macrophages with GM-CSF induced expression of the CD11b surface adhesion molecule, which was associated with increased attachment to tumor cells. All KM12SM cells were sensitive to macrophage-mediated lysis in the presence of rGM-CSF and recombinant MCP-1. Collectively, the results demonstrate that tumor cell-derived GM-CSF stimulates PMN and macrophages to secrete macrophage-inflammatory protein-1alpha and MCP-1, which triggers recruitment of mononuclear cells, induces expression of adhesion molecules on macrophages, and enhances contact-dependent cytolysis of tumor cells.     

The role of proteases in fibronectin matrix remodeling in thyroid epithelial cell monolayer cultures

Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence. In non-confluent cells FN forms a fibrillar array associated with the ventral cell surface. However, basal FN is progressively removed in confluent cultures and substituted by non-fibrillar FN deposits at lateral cell domains in regions of cell-cell contacts. FRT cells secrete and expose on the plasma membrane the tissue-type plasminogen activator and, in serum-free cultures, plasminogen induces a rapid loss of FN fibrils. Incubation with plasmin inhibitors greatly reduces this effect. FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment. This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are pre-incubated with carboxypeptidase B, which prevents plasminogen binding to the cells. A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence. These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmin-dependent (tPA activated) and plasmin-independent proteolytic activities are involved.     

Fibronectin enhances viability and alters cytoskeletal functions (with effects on the phosphatidylinositol 3-kinase pathway) in small cell lung cancer

Small cell lung cancer (SCLC) is a rapidly progressive disease with ultimate poor outcome. SCLC has been shown to interact closely with the stromal and extracellular matrix (ECM) components of the diseased host. ECM consists of type I/IV collagen, laminin, vitronectin, and fibronectin (FN) among others. Herein, we investigated the behavior of a SCLC cell line (NCI-H446) on FN-coated surface. Over a course of 72 h, FN (10 micro g/ml) caused both increased survival and proliferation of NCI-H446 cells. Survival under serum-starved conditions increased 1.44-fold and proliferation in the presence of fetal calf serum increased by 1.30-fold. The phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 reduced both survival and proliferation of NCI-H446 cells (0.48- and 0.27-fold, respectively), even on FN-coated surface. We next determined the effects of FN on cytoskeletal function such as cell motility/morphology and adhesion. Over a course of 24 h, FN reduced aggregation of NCI-H446 cells and induced flattened cellular morphology with neurite-like projections after 1 h, however, in the presence of LY294002, the cells rounded up. Adhesion of NCI-H446 cells also increased with FN (4.47-fold) which was abrogated with LY294002 treatment. This correlated with phosphorylation of the cytoskeletal protein p125FAK, on Tyr397, Tyr861 and Ser843 residues with FN. Even in the presence of LY294002, these serine/tyrosine residues were still phosphorylated on FN-coated surface. In contrast, the focal adhesion protein paxillin was not phosphorylated at Tyr31 with FN. In summary, FN stimulation of SCLC cells leads to enhancement of viability and changes in cytoskeletal function that are partially mediated through the PI3-K pathway.     

Functional differences between integrin alpha4 and integrins alpha5/alphav in modulating the motility of human oral squamous carcinoma cells in response to the V region and heparin-binding domain of fibronectin

The high-affinity heparin-binding domain and the V region of fibronectin (FN) mediate invasion and migration of human oral squamous cell carcinoma (SCC) cells. We investigated the role of the alpha4, alpha5, and alphav integrin receptors--which are central to mediating interactions with these domains of FN--in regulating SCC cell migration. SCC cells expressed the alpha4, alpha5, and alphav integrin subunits on their surface, although alpha4 expression was low. Treatment with recombinant FN proteins containing an alternatively spliced V region (V+) and either an unmutated (H+) or a mutated, nonfunctional high-affinity heparin-binding domain (H-) increased expression of alpha5 and alphav and cell motility. Antisense alpha5 or alphav oligonucleotides inhibited cell motility stimulated by FN proteins, as did blocking antibodies to alpha5 or alphav. Blocking antibodies to alpha5 increased alphav and alpha4 levels, and blocking antibodies to alphav increased the levels of alpha5 and alpha4, without increasing cell motility. In contrast, an antisense alpha4 oligonucleotide and alpha4-blocking antibodies increased cell motility, especially migration stimulated by V+H+ and V+H- FN proteins. alpha4-Blocking antibodies alone increased motility, probably by inducing alpha5 and alphav expression. Transfection with alpha4 cDNA decreased cell motility and alpha5 and alphav expression. Thus, the increased motility induced by the FN protein is probably mediated by alphav and alpha5, whereas alpha4 downregulates this process in a transdominant fashion.     

Fibronectin gene expression in proliferating, quiescent, and SV40-infected mouse kidney cells

To study alterations in cellular gene expression in mouse kidney cell cultures infected with simian virus 40 (SV40) or polyomavirus, we performed a differential screening of a mouse kidney cDNA library with probes prepared from mRNAs of virus-infected and mock-infected cells. We isolated and characterized cDNA recombinant pKT13 which detected increased mRNA levels in infected cells. Sequence analysis of pKT13 revealed close to 100% homology with the 3'-end of mouse fibronectin (FN) mRNA. Since primary cultures of baby mouse kidney cells have been extensively characterized in our laboratories, we studied FN gene expression at different stages of uninfected and virus-infected cultures. High levels of FN and of its mRNA were found in the kidneys of suckling mice, while in primary cultures of proliferating epithelial kidney cells the expression of FN was very low until the cultures became confluent. Thereafter FN increased and reached high levels in cells which were irreversibly arrested in phase Go and which had apparently exhausted their finite division potential. Infection of confluent cultures with polyomavirus or SV40 resulted in a further stimulation of FN gene expression. However, during abortive infection with SV40, FN mRNA and FN levels decreased with emergence of transformed cells and were low in an established SV40-transformed mouse kidney cell line. These changes in FN gene expression suggest that high levels of FN might be indicative in vivo for terminal differentiation and in vitro for cellular senescence.     

Heterologous exchanges of the glycoprotein and the matrix protein in a Novirhabdovirus

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are two salmonid rhabdoviruses replicating at low temperatures (14 to 20 degrees C). Both viruses belong to the Novirhabdovirus genus, but they are only distantly related and do not cross antigenically. By using a recently developed reverse-genetic system based on IHNV (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000), we investigated the ability to exchange IHNV glycoprotein G with that of VHSV. Thus, the IHNV genome was modified so that the VHSV G gene replaced the complete IHNV G gene. A recombinant virus expressing VHSV G instead of IHNV G, rIHNV-Gvhsv, was generated and was shown to replicate as well as the wild-type rIHNV in cell culture. This study was extended by exchanging IHNV G with that of a fish vesiculovirus able to replicate at high temperatures (up to 28 degrees C), the spring viremia of carp virus (SVCV). rIHNV-Gsvcv was successfully recovered; however, its growth was restricted to 14 to 20 degrees C. These results show the nonspecific sequence requirement for the insertion of heterologous glycoproteins into IHNV virions and also demonstrate that an IHNV protein other than the G protein is responsible for the low-temperature restriction on growth. To determine to what extent the matrix (M) protein interacts with G, a series of chimeric pIHNV constructs in which all or part of the M gene was replaced with the VHSV counterpart was engineered and used to recover the respective recombinant viruses. Despite the very low percentage (38%) of amino acid identity between the IHNV and VHSV matrix proteins, viable chimeric IHNVs, harboring either the matrix protein or both the glycoprotein and the matrix protein from VHSV, were recovered and propagated. Altogether, these data show the extreme flexibility of IHNV to accommodate heterologous structural proteins.     

Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) are rhabdoviruses that infect salmonids, producing serious economic losses. Two recombinant IHN viruses were generated by reverse genetics. For one (rIHNV GFP) the IHNV NV gene was replaced with the green fluorescent protein (GFP) gene. In the other (rIHNV-Gvhsv GFP) the G gene was also exchanged for that of VHSV. No mortalities, external signs or histological lesions were observed in experimental infections conducted with the recombinant viruses. Neither the rIHNV GFP nor rIHNV-Gvhsv GFP was detected by RT-PCR in any of the examined tissues from experimentally infected fish. In order to assess their potential as vaccines against the wild type viruses, rainbow trout were vaccinated with the recombinant viruses by intraperitoneal injection and challenged 30 d later with virulent IHNV or VHSV. The GFP viruses provided protection against both wild type viruses. None of the recombinant viruses induced antibody production, and the expression of interferon (IFNalpha4) and interferon induced genes such as Mx protein and ISG-15 was not different to that of controls. The rIHNV-Gvhsv GFP did not inhibit cellular apoptosis as it was observed in an IHNV inoculated fish cell line. These studies suggest that the recombinant rIHNV-Gvhsv GFP is a promising candidate as a live recombinant vaccine and also provides a good model to further study viral pathogenicity and the molecular basis of protection against these viral infections.     

Influences of the recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells in serum-free culture

To improve the safety of cellular therapy products, it is necessary to establish a serum-free cell culture method that can exclude animal-derived materials in order to avoid contamination with transmissible agents. It would be optimal if the proteins necessary to a serum-free culture could be provided as recombinant proteins. In this study, the influences of recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells (HUVECs) in serum-free culture were examined in comparison with the influence of plasma fibronectin (FN). The recombinant proteins used were Pronectin F (PF), Pronectin F PLUS (PFP), Pronectin L (PL), Retronectin (RN), and Attachin (AN). HUVECs adhered more efficiently on PF or PFP than on FN. No cells adhered on PL. Regarding the VEGF or bFGF-induced cell growth, the cells on PF and PFP proliferated at a similar rate to the cells on FN. RN and AN were less effective in supporting cell growth. Since cell adhesion on PF and PFP induced phosphorylation of focal adhesion kinase, they are thought to activate integrin-mediated intracellular signaling. The cells cultured on PF or PFP were able to produce prostaglandin I(2) or tissue-plasminogen activator in response to thrombin. However, thrombin caused detachment of the cells from PF but not from PFP or FN, meaning that the cells were able to adhere more tightly on PFP or FN than on PF. These data indicate that PFP could be applicable as a substitute for plasma FN.