The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice

Real-time PCR and fluorogenic probes were combined in a simple, rapid and sensitive method to genotype murine breeding stocks and their progeny for a point mutation. DNA from tail biopsies of newborn mice was mixed with amplification primers and fluorogenic hybridization probes in a PCR mixture. The primers were designed to amplify a region of the Fas-Ligand gene including the site for the gld natural point mutation. The fluorogenic hybridization probes overlaid this target sequence and were used to detect amplification of the PCR fragment as well as determine the presence of the point mutation using fluorescence resonance energy transfer (FRET). Both mutated and wild-type forms of the gene fragment were amplified as detected with real-time PCR. Melting curve profiles completed on each amplified sample revealed the genotype for each mouse. These genotypes were confirmed by sequencing the amplified fragments. These results suggest real-time spectrofluorometric PCR techniques incorporating FRET-based hybridization probes may be used for rapid, sensitive, inexpensive and reliable genotyping.     

Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9.

The nucleotide sequence coding for the cytotoxic T-lymphocyte (CTL) protein perforin 1 (P1) has been determined and the corresponding protein sequence has been derived. Murine CTL cDNA libraries contained in the vector lambda gt11 were screened by using a monospecific antiserum to purified P1. Three recombinant phages were isolated and their cDNA inserts were sequenced. The derived protein sequence contains 554 amino acids and displays, as expected, considerable homology with certain functional domains in the complement components C9, C8 alpha, C8 beta, and C7. The identity of P1 cDNA clones was verified by prokaryotic expression and the reactivities of antisera produced to the expressed proteins. In addition, antisera were produced to two synthetic peptides located in the center and C-terminal portions of P1. All antisera reacted with purified P1. In Northern blot analyses, P1 cDNA probes recognized a 2.9-kilobase mRNA only in CTL. Perforin mRNA was found in all cloned CTL and in all mixed lymphocyte reactions that gave rise to cytotoxic cells. Perforin mRNA was also detected in virus-specific CTL that had been generated in vivo and isolated from liver tissue of mice infected with lymphocytic choriomeningitis virus. The cell-specific expression of perforin is consistent with its postulated role in cytolysis.     

Secreted heat shock protein gp96-Ig: next-generation vaccines for cancer and infectious diseases

Over the past decade, our laboratory has developed a secreted heat shock protein (HSP), chaperone gp96, cell-based vaccine that generates effective anti-tumor and anti-infectious immunity in vivo. Gp96-peptide complexes were identified as an extremely efficient stimulator of MHC I-mediated antigen cross-presentation, generating CD8 cytotoxic T-lymphocyte responses detectable in blood, spleen, gut and reproductive tract to femto-molar concentrations of antigen. These studies provided the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce both systemic and mucosal immunity. This approach takes advantage of the combined adjuvant and antigen delivery capacity of gp96 for the generation of cytotoxic immunity against a wide range of antigens in both anti-vial and anti-cancer vaccination. Here, we review the vaccine design that utilizes the unique property/ability of endoplasmic HSP gp96 to bind antigenic peptides and deliver them to antigen-presenting cells.

     

Decidual macrophages are the population of decidual adherent cells which regulates perforin expression in cytolytic cells.

PROBLEM: We have shown that addition of decidual adherent cells (DAC) to the culture of decidual lymphocytes (DL) prevents the downregulation of perforin expression in these cells. Because DAC are a mixture of various cell populations, the aim is to analyze immunophenotypic characteristics of DAC and to determine which cell population is involved in the regulation of perforin expression. METHOD OF STUDY: First trimester pregnancy decidual cells were obtained by enzymatic tissue digestion. Decidual cells and peripheral blood lymphocytes (PBL) were centrifuged on Ficoll-Hypaque density gradient and cultured overnight to obtain adherent cells, which were analyzed by flow cytometry and immunocytochemically. RESULTS: Almost all peripheral blood adherent cells (PBAC) (ca 90%) expressed monocyte/macrophage markers but only 10-20% of DAC. The rest of DAC expressed markers of stromal cells. HLA-DR depleted population of DAC (stromal cells only) could not prevent downregulation of perforin expression in cultured DL and PBL. CONCLUSION: Decidual macrophages are involved in the regulation of perforin expression in DL.     

Assessment of perforin expression in peripheral blood lymphocytes in psoriatic patients during exacerbation of disease

There are very few data concerning the role played by cell-mediated cytotoxicity, particularly at the molecular level, in the course of psoriasis. Both cytotoxic T lymphocytes (CTL) and natural killer cells contain in their granules the cytolytic protein perforin, a mediator in cell-mediated cytotoxicity reactions. The aim of this study was to analyze perforin expression in various sets and subsets of perforin-positive peripheral blood lymphocytes in 17 patients with chronic psoriasis vulgaris in the exacerbation phase. The results were compared with those of an age- and sex-matched healthy control group (n = 21). Perforin (intracellular antigen) and cell surface antigens were detected using the simultaneous double-staining method. We found a significant increase in perforin (P) expression in the patient group for CTL (CD3+P+ cells), which are located mostly in the CD8+ population of T lymphocytes (CD8+P+).     

Nucleotide sequence of cDNA and derived amino acid sequence of human complement component C9.

The nucleotide sequence coding for the ninth component of human complement (C9) has been determined and the corresponding amino acid sequence has been derived. A human liver cDNA library was screened by the colony-hybridization technique using two radiolabeled oligonucleotide probes that correspond to known regions of the C9 amino acid sequence. Two recombinant plasmids were isolated and their cDNA inserts were sequenced. The derived protein sequence consists of 537 amino acids in a single polypeptide chain. A profile of the hydropathic index versus sequence number indicates that the amino-terminal half of C9 is predominantly hydrophilic in character whereas the carboxyl-terminal section of this protein is more hydrophobic. The amphipathic organization of the primary structure of C9 is consistent with the known potential of polymerized C9 to penetrate lipid bilayers, causing the formation of transmembrane channels.     

Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation, which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo, antibody blockade of TNFSF15 (TL1A), which is the ligand for TNFR25, inhibits lung inflammation and production of Th2 cytokines such as IL-13, even when administered days after airway antigen exposure. Similarly, blockade of TNFR25 by a dominant-negative (DN) transgene, DN TNFR25, confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant, NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells, but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung, and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics.     

Modulation of perforin expression in the decidual and peripheral blood cytotoxic lymphocytes in culture.

PROBLEM: Perforin (P) is a cytolytic molecule located in intracellular granules of cytotoxic lymphocytes both in the peripheral blood and decidua of pregnancy. The aim was to analyze the kinetics of P expression during in vitro culture and modulation of P expression by adherent cells, their supernatants and mitogen (PHA) stimulation. METHOD OF STUDY: P (intracellular antigen) was detected by flow cytometry in the suspension of first trimester pregnancy peripheral blood lymphocytes (PBL) and decidual lymphocytes (DL). RESULTS: A decrease of the percentage of P+ cells was obtained after 1 hr incubation and was prevented by addition of 30% of decidual adherent cells (DAC) or their supernatants. Upregulation of P expression was obtained when, in addition to adherent cells, DL and PBL were stimulated by PHA. DAC present in the culture in physiological concentrations prevent downregulation of P expression. CONCLUSION: DAC located in the vicinity of decidual cytotoxic lymphocytes, owing to their unique ability to produce a wide range of substances on demand, contribute to the high level of P expression in the decidua of pregnancy.     

Present and future of lung cancer vaccines

New approaches are needed to improve the current treatment of lung cancer. Inducing an immune response against lung tumour cells with vaccines represents an attractive therapy. However, lung tumours had not been considered good targets for vaccine therapy and, therefore, immune approaches have not been studied extensively in this setting. Current experimental strategies for antitumour vaccines include the generation of active immune responses against specific tumour antigens. Understanding the mechanisms of antitumour immunity and identifying relevant tumour-specific antigens will probably improve therapeutic strategies and provide avenues for the future of lung cancer therapy. There have been a number of preclinical immunotherapy trials suggesting activity, and a smaller number of human clinical trials using various vaccines in lung cancer. Initial data from these trials have shown preliminary evidence of induction of immune responses and suggest clinical activity. This paper reviews some of the most important developments in vaccines for lung cancer.