Human articular chondrocytes immortalized by HPV-16 E6 and E7 genes: Maintenance of differentiated phenotype under defined culture conditions

AbstractObjective To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases.Design Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification.Results Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes.Conclusions The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans

Co-occurrence of two cases of progressive multifocal leukoencephalopathy in a natalizumab ``infusion group''

We observed two cases of progressive multifocal leukoencephalopathy (PML) that occurred in the same infusion group. The group consisted of four patients with relapsing-remitting multiple sclerosis (RRMS) who had been treated with natalizumab (NAT) in the same medical practice for more than four years at the same times and in the same room, raising concerns about viral transmission between members of the infusion group. DNA amplification and sequence comparison of the non-coding control region (NCCR) of JC virus (JCV) present in cerebrospinal fluid (CSF) samples from PML patients #1 and #2 revealed that the amplified JCV sequences differed from the JCV archetype. The NCRR of the viral DNA was unique to each patient, arguing against the possibility of viral transmission between patients. Statistical considerations predict that similar co-occurrences of PML are likely to happen in the future

Co-occurrence of two cases of progressive multifocal leukoencephalopathy in a natalizumab ``infusion group''

We observed two cases of progressive multifocal leukoencephalopathy (PML) that occurred in the same infusion group. The group consisted of four patients with relapsing-remitting multiple sclerosis (RRMS) who had been treated with natalizumab (NAT) in the same medical practice for more than four years at the same times and in the same room, raising concerns about viral transmission between members of the infusion group. DNA amplification and sequence comparison of the non-coding control region (NCCR) of JC virus (JCV) present in cerebrospinal fluid (CSF) samples from PML patients #1 and #2 revealed that the amplified JCV sequences differed from the JCV archetype. The NCRR of the viral DNA was unique to each patient, arguing against the possibility of viral transmission between patients. Statistical considerations predict that similar co-occurrences of PML are likely to happen in the future

Changes in JC virus-specific T cell responses during natalizumab treatment and in natalizumab-associated progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) induced by JC virus (JCV) is a risk for natalizumab-treated multiple sclerosis (MS) patients. Here we characterize the JCV-specific T cell responses in healthy donors and natalizumab-treated MS patients to reveal functional differences that may account for the development of natalizumab-associated PML. CD4 and CD8 T cell responses specific for all JCV proteins were readily identified in MS patients and healthy volunteers. The magnitude and quality of responses to JCV and cytomegalovirus (CMV) did not change from baseline through several months of natalizumab therapy. However, the frequency of T cells producing IL-10 upon mitogenic stimulation transiently increased after the first dose. In addition, MS patients with natalizumab-associated PML were distinguished from all other subjects in that they either had no detectable JCV-specific T cell response or had JCV-specific CD4 T cell responses uniquely dominated by IL-10 production. Additionally, IL-10 levels were higher in the CSF of individuals with recently diagnosed PML. Thus, natalizumab-treated MS patients with PML have absent or aberrant JCV-specific T cell responses compared with non-PML patients, and changes in T cell-mediated control of JCV replication may be a risk factor for developing PML. Our data suggest further approaches to improved monitoring, treatment and prevention of PML in natalizumab-treated patients

Constraints on the χ_(c1) versus χ_(c2) polarizations in proton-proton collisions at √s = 8 TeV

The polarizations of promptly produced χ_(c1) and χ_(c2) mesons are studied using data collected by the CMS experiment at the LHC, in proton-proton collisions at √s=8  TeV. The χ_c states are reconstructed via their radiative decays χ_c → J/ψγ, with the photons being measured through conversions to e⁺e⁻, which allows the two states to be well resolved. The polarizations are measured in the helicity frame, through the analysis of the χ_(c2) to χ_(c1) yield ratio as a function of the polar or azimuthal angle of the positive muon emitted in the J/ψ → μ⁺μ⁻ decay, in three bins of J/ψ transverse momentum. While no differences are seen between the two states in terms of azimuthal decay angle distributions, they are observed to have significantly different polar anisotropies. The measurement favors a scenario where at least one of the two states is strongly polarized along the helicity quantization axis, in agreement with nonrelativistic quantum chromodynamics predictions. This is the first measurement of significantly polarized quarkonia produced at high transverse momentum