Chondrogenic potential of human dermal fibroblasts in a contractile, soft, self-assembling, peptide hydrogel.

The present paper describes a simple approach to obtain three-dimensional (3D) cartilage constructs using human normal dermal fibroblasts (hNDFs) cultured in a self-assembling peptide nanofibre scaffold. During the first days of culture, the 3D constructs underwent morphological changes consisting of a substantial contraction process that ended in a small compact structure. During this process the system became sensitive to induction with standard chondrogenic medium, evidenced by the expression of specific markers of mature cartilage. First, it was detected that the samples become highly stained with toluidine blue dye over time (40-50 days), indicating that the system produced significantly high amounts of glycosaminoglycans. By quantitative PCR, it was confirmed that the system significantly upregulated the expression of the proteoglycan aggrecan, a good indicator of cartilage commitment. Moreover, collagen type II was upregulated at protein level, confirming that the system differentiated to a chondrocyte-like construct. Additionally, during the first days of culture in control medium analysed hNDFs proliferation capacity in this 3D system was analysed. This platform could be used in the future to obtain an autologous source of cells from a simple patient skin biopsy, which could be easily translated into a low-cost and effective regenerative therapy.

High Inter-Individual Diversity of Point Mutations, Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells.

The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4% in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.

Loss of HIV-specific memory B-cells as a potential mechanism for the dysfunction of the humoral immune response against HIV.

A central, yet unresolved issue in the pathogenesis of HIV disease is the mechanism of antibody perturbation. In this study, HIV-specific memory B-cells were quantified in groups of infected subjects and compared with memory responses to other antigens and antibody titers. HIV-specific memory B-cell responses were vigorous in individuals with CD4(+) T-cell counts >350/microl and weak or undetectable in subjects with CD4(+) T-cell numbers <200/microl. Memory B-cell loss was permanent, because antiretroviral therapy failed to restore HIV-specific memory responses while influenza- and tetanus toxoid-specific memory B-cells remained unaffected or recovered. Antibody titers to Gag strongly correlated with memory B-cell frequencies. In contrast, Env-specific antibodies were maintained in advanced disease despite low or undetectable levels of memory B-cells. These results provide a potential mechanism by which destruction of HIV-specific CD4(+) T-cells affects the humoral immune response against HIV and compromises the ability to maintain an effective antibody response.

Antibody-mediated binding of fluorescent HIV Gag and influenza nucleoprotein tetramers to blood cells.

Antibody-mediated elimination of foreign antigens contributes to immune protection from viral infection. We have generated phycoerythrin-conjugated HIV-1 Gag/p24 and influenza virus nucleoprotein tetramers for flow cytometric analysis of blood cell populations involved in antibody-mediated immunity. We show that in the presence of antigen-specific antibodies fluorescent antigen tetramers bound to different blood cell populations including granulocytes, monocytes and lymphocytes. Binding to B-lymphocytes was particularly efficient. The interaction was primarily mediated by complement. Fcgamma receptor-mediated antigen binding by B-cells was significantly less effective. The study shows that fluorescent antigen tetramers are useful to quantify cell populations involved in complement- and Fcgamma-mediated immune responses in viral infections.

Peptides from the SARS-associated coronavirus as tags for protein expression and purification.

Protein tagging with a peptide is a commonly used technique to facilitate protein detection and to carry out protein purification. Flexibility with respect to the peptide tag is essential since no single tag suites all purposes. This report describes the usage of two short peptides from the SARS-associated coronavirus nucleocapsid (SARS-N) protein as protein tags. Plasmids for the generation of tagged proteins were generated by ligating synthetic oligonucleotides for the peptide-coding regions downstream of the protein coding sequence. The data show recognition of prokaryotically expressed HIV-1 Gag/p24 fusion protein by Western blot and efficient affinity purification using monoclonal antibodies against the tags. The SARS peptide antibody system described presents an alternative tagging opportunity in the growing field of protein science.

Antigenic and cellular localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using monoclonal antibodies.

A member of the family of coronaviruses has previously been identified as the cause of the severe acute respiratory syndrome (SARS). In this study, several monoclonal antibodies against the nucleocapsid protein have been generated to examine distribution of the nucleocapsid in virus-infected cells and to study antigenic regions of the protein. Confocal microscopic analysis identified nucleocapsids packaged in vesicles in the perinuclear area indicating viral synthesis at the endoplasmic reticulum and Golgi apparatus. The monoclonal antibodies bound to the central and carboxyterminal half of the nucleocapsid protein indicating prominent exposure and immunogenicity of this part of the protein. Antibodies recognised both linear and conformational epitopes. Predictions of antigenicity using mathematical modelling based on hydrophobicity analysis of SARS nucleoprotein could not be confirmed fully. Antibody binding to discontinuous peptides provides evidence that amino acids 274-283 and 373-382 assemble to a structural unit particularly rich in basic amino acids. In addition, amino acids 286-295, 316-325 and 361-367 that represent the epitope recognised by monoclonal antibody 6D11C1 converge indicating a well-structured C-terminal region of the SARS virus nucleocapsid protein and functional relationship of the peptide regions involved. Alternatively, dimerisation of the nucleocapsid protein may result in juxtaposition of the amino acid sequences 316-325 and 361-367 on one nucleoprotein molecule to amino acid 286-295 on the second peptide. The monoclonal antibodies will be available to assess antigenicity and immunological variabilities between different SARS CoV strains.