[An experimental study on the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts].

OBJECTIVE: To investigate the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts. METHODS: Eukaryotic expression vector pcDNA3-p16 was constructed and imported into KFb by gene transfection mediated by liposome. And the positive clones were screened by G418. The transfected and untransfected KFbs were stained by Immunocytochemical method. The expression of p16 protein was observed. The changes of the proliferation and DNA synthesis of KFb before and after transfection were observed and compared by drafting cell growth curve and by (3)H-TdR incorporation method. RESULTS: The recombinant vector pcDNA3-p16 was successfully constructed and identified by enzyme digestion. The positive clones were identified by G418 selection for 10 days from transfected KFb and with p16 protein expression. The growth rate of transfected KFb slowed down obviously and its DNA synthesis decreased significantly (P < 0.05) when compared with those of normal KFb. CONCLUSION: p16 gene might inhibit the growth and DNA synthesis of KFb.

CC chemokine receptor 7-dependent and -independent pathways for lymphocyte homing: modulation by FTY720.

Cognate interaction of chemokine receptor CCR7 on lymphocytes with its ligands CCL19 and CCL21 expressed on high endothelial venules (HEVs) is essential for effective migration of T and B cells across HEVs into secondary lymphoid organs. Plt mice, which lack expression of CCL19 and CCL21-ser, both ligands for CCR7 on HEVs, as well as CCR7-deficient mice, have a defective cell migration and reduced homing of lymphocytes. FTY720, a novel immunosuppressant, causes a reduction of lymphocytes in peripheral blood and tissues and their sequestration into lymphoid tissues. In this study we demonstrate that FTY720 rescues the homing defect in both CCR7(-/-) mice and plt mice. After FTY720 treatment, the number of CD4(+) and CD8(+) T cells as well as B cells in peripheral blood is reduced while pertussis toxin-sensitive homing into peripheral lymph nodes, mesenteric lymph node, and Peyer's patches is increased. Immunohistology demonstrates that FTY720 enables these cells to enter lymphoid tissue through HEVs. Thus, our data suggest an alternative G-alpha(i)-dependent, CCR7-CCL19/CCL21-independent mechanism for lymphocyte homing through HEVs which is strongly augmented in the presence of FTY720.

Dendritic cells in autoimmune diseases.

Subclinical autoimmune responses can be frequently detected in healthy individuals. Sustained activation of autoreactive lymphocytes is, however, required for the development of autoimmune diseases associated with ongoing tissue destruction either in single organs or generalized with multiple manifestations. Clinical and experimental evidence suggests that prolonged presentation of self antigens by dendritic cells is crucial for the development of destructive autoimmune disease. We discuss here a simplified threshold model where the key parameters for the magnitude of the autoimmune response are the amount of previously ignored self peptides presented by dendritic cells and the duration of the antigen presentation in secondary lymphoid organs. Multiple factors influence the threshold for the conversion of an autoimmune response to overt autoimmune disease. Frequent or persistent viral infections of the target organ may favor autoimmune disease by increasing the amounts of released self antigens, generating cytokine-mediated bystander activation of self-reactive lymphocytes and/or sustaining a chronic response via neoformation of lymphoid structures in the target organ.

CD4(+) T cell subsets during virus infection. Protective capacity depends on effector cytokine secretion and on migratory capability.

To analyze the antiviral protective capacities of CD4(+) T helper (Th) cell subsets, we used transgenic T cells expressing an I-A(b)-restricted T cell receptor specific for an epitope of vesicular stomatitis virus glycoprotein (VSV-G). After polarization into Th1 or Th2 effectors and adoptive transfer into T cell-deficient recipients, protective capacities were assessed after infection with different types of viruses expressing the VSV-G. Both Th1 and Th2 CD4(+) T cells could transfer protection against systemic VSV infection, by stimulating the production of neutralizing immunoglobulin G antibodies. However, only Th1 CD4(+) T cells were able to mediate protection against infection with recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G). Similarly, only Th1 CD4(+) T cells were able to rapidly eradicate Vacc-IND-G from peripheral organs, to mediate delayed-type hypersensitivity responses against VSV-G and to protect against lethal intranasal infection with VSV. Protective capacity correlated with the ability of Th1 CD4(+) T cells to rapidly migrate to peripheral inflammatory sites in vivo and to respond to inflammatory chemokines that were induced after virus infection of peripheral tissues. Therefore, the antiviral protective capacity of a given CD4(+) T cell is governed by the effector cytokines it produces and by its migratory capability.

Antiviral protection after DNA vaccination is short lived and not enhanced by CpG DNA.

In this study, we investigated the potential of a DNA vaccine expressing the minimal cytotoxic T lymphocyte (CTL) epitope gp33 of the lymphocytic choriomeningitis virus glycoprotein to protect against infection of a non-lymphoid organ and compared this to protection against a systemic infection. Furthermore, since immune stimulatory sequences have been shown to augment CTL responses, we examined the capacity of CpG DNA to enhance CTL memory. The data show that DNA vaccination with a gp33-based gene construct induced short-lived gp33-specific CTL which protected against a systemic infection but not against a peripheral infection. Immune stimulatory sequences were incapable of either prolonging CTL memory or promoting protection against infection of a peripheral organ.

Determination of natural versus laboratory human infection with Mayaro virus by molecular analysis.

A laboratory worker developed clinical signs of infection with Mayaro virus (Togaviridae), an arbovirus of South and Central America, 6 days after preparation of Mayaro viral antigen and 10 days after a trip to a rain forest. There was no evidence of skin lesions during the antigen preparation, and level 3 containment safety measures were followed. Therefore, molecular characterization of the virus was undertaken to identify the source of infection. RT-PCR and DNA sequence comparisons proved the infection was with the laboratory strain. Airborne Mayaro virus contamination is thus a hazard to laboratory personnel.

Determination of T cell epitopes with random peptide libraries.

A new approach to T cell epitope determination is presented. Critical amino acids for the induction of cytotoxic T cell responses were identified using synthetic peptide libraries with single defined sequence positions combined with randomized sequence positions. Sequences for potential T cell epitopes were deduced from scan profiles using combinations of the active amino acids. Highly potent epitopes for cytotoxic T lymphocytes were obtained. Epitopes defined by this approach are, as shown in this communication, not necessarily the natural epitopes and, therefore, were named synthetic epitopes. They can serve effectively for the development of vaccines or for the determination of T cell receptor antagonists.

Specificity and degeneracy of minor histocompatibility antigen-specific MHC-restricted CTL.

Random peptide libraries were employed to investigate the specificity of Ag recognition by H-3-specific, H-2K(b)-restricted CTL clones. The peptide libraries consist of octapeptides with one defined sequence position and mixtures of 19 amino acids (all proteinogenic amino acids except for cysteine) in the remaining seven sequence positions. The complete set of 152 peptide libraries includes all octapeptides possible with these amino acids. Responses of the CTL clones to these peptide libraries reveal patterns of preferred epitope amino acids. Depending on the CTL clone tested, varying numbers of different amino acids were identified for the different sequence positions indicating degeneracy of Ag recognition. Sequences for synthetic epitopes active at low pM concentrations could be deduced from these patterns. They confirm that TCRs of CTL clones do not exhibit specificity for unique ligand structures but rather can interact with sets of ligands. The sequences of peptides recognized by a single clone exhibit great sequence heterogeneity.