Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Introduction

The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.

Methods

Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.

Results

Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.

Conclusions

Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.     

Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells

After attachment to receptors, reovirus virions are internalized by endocytosis and exposed to acid-dependent proteases that catalyze viral disassembly. Previous studies using the cysteine protease inhibitor E64 and a mutant cell line that does not support reovirus disassembly suggest a requirement for specific endocytic proteases in reovirus entry. This study identifies the endocytic proteases that mediate reovirus disassembly in murine fibroblast cells. Infection of both L929 cells treated with the cathepsin L inhibitor Z-Phe-Tyr(t-Bu)-diazomethyl ketone and cathepsin L-deficient mouse embryo fibroblasts resulted in inefficient proteolytic disassembly of viral outer-capsid proteins and decreased viral yields. In contrast, both L929 cells treated with the cathepsin B inhibitor CA-074Me and cathepsin B-deficient mouse embryo fibroblasts support reovirus disassembly and growth. However, removal of both cathepsin B and cathepsin L activity completely abrogates disassembly and growth of reovirus. Concordantly, cathepsin L mediates reovirus disassembly more efficiently than cathepsin B in vitro. These results demonstrate that either cathepsin L or cathepsin B is required for reovirus entry into murine fibroblasts and indicate that cathepsin L is the primary mediator of reovirus disassembly. Moreover, these findings suggest that specific endocytic proteases can determine host cell susceptibility to infection by intracellular pathogens.     

A Monoclonal Antibody Specific for Reovirus Outer-Capsid Protein ς3 Inhibits ς1-Mediated Hemagglutination by Steric Hindrance

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 microg per ml, whereas that of Fabs was >200 microg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma3 and mu1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.     

The human papillomavirus 16 E6 protein binds to tumor necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis

High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that transfection of HPV 16 E6 into the tumor necrosis factor (TNF)-sensitive LM cell line protects expressing cells from TNF-induced apoptosis in a p53-independent manner, and the purpose of this study was to determine the molecular mechanism underlying this protection. Caspase 3 and caspase 8 activation were significantly reduced in E6-expressing cells, indicating that E6 acts early in the TNF apoptotic pathway. In fact, E6 binds directly to TNF R1, as shown both by co-immunoprecipitation and mammalian two-hybrid approaches. E6 requires the same C-terminal portion of TNF R1 for binding as does TNF R1-associated death domain, and TNF R1/TNF R1-associated death domain interactions are decreased in the presence of E6. HA-E6 also blocked cell death triggered by transfection of the death domain of TNF R1. Together, these results provide strong support for a model in which HPV E6 binding to TNF R1 interferes with formation of the death-inducing signaling complex and thus with transduction of proapoptotic signals. They also demonstrate that HPV, like several other viruses, has developed a method for evading the TNF-mediated host immune response.     

Prolonged replication in the mouse central nervous system of reoviruses isolated from persistently infected cell cultures.

We examined pathogenic characteristics of plaque-purified reoviruses isolated from persistently infected L-cell cultures (PI viruses) after intracranial inoculation into newborn mice. The PI viruses were isolated from independent cultures initiated with high-passage stocks of the wild-type (wt) strain, type 3 Dearing. The virulence of most PI viruses was equivalent to that of the wt strain. However, replication of PI viruses in the central nervous system of infected mice was prolonged to 25 (but not 50) days postinoculation. Thirty-eight percent (n = 186) of mice inoculated with the PI viruses had residual virus detectable in brain tissue 25 days after inoculation, in contrast to only 16% (n = 57) of mice inoculated with wt virus (P = 0.009). Mean residual brain titers were more than 20-fold higher in mice inoculated with PI viruses compared with wt virus (4.3 x 10(4) versus 2.1 x 10(3); P = 0.006). Tropism of PI virus within the brain resembled that of wt virus, and the distribution of PI virus antigen in the brain did not change over time. The extent of necrosis in the brains of mice harboring PI virus 25 days after inoculation was minimal, despite continued presence of high titers of infectious virus. The latter observation resembles the absence of cytopathicity seen in L-cell cultures persistently infected with reovirus. These observations suggest that the interaction of PI viruses with cells can be altered in vivo as well as in cell culture, but virus is eventually cleared from the infected animal.