Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress

Sphingosine, which is on the pathway of sphingomyelin degradation, activates phospholipase C (PLC) delta1 moderately. In the liposome assay effect of sphingosine on PLC delta1 activity depends on KCl concentration. Stimulation of PLC delta1 by sphingosine increased as the KCl concentration is increased from 0 to 100 mM, and then diminished with the increasing KCl. In the liposome assay sphingosine diminishes inhibition of PLC delta1 by sphingomyelin. To determine the domain of PLC delta1 which interacts with sphingosine active proteolytic fragments of PLC delta1 were generated by trypsin digestion of the native enzyme. Sphingosine affects the activity of PLC delta1 fragment which lacked the amino-terminal domain (first 60 amino acids) but not the active fragment that has cleaved the domain spanning the X and Y region of PLC delta1. These observations indicate that for interaction of sphingosine with PLC delta1 intact domain that span regions of conservation, designated as X and Y is necessary. When the activity of PLC delta1 was assayed with PIP2 in the erythrocyte membrane as substrate, sphingosine strongly inhibited PLC delta1. The other homolog of sphingosine 4-hydroxysphinganine (phytosphingosine) inhibited PLC delta1 to much lesser extent. The activity of PLC delta1 was inhibited by 68% and 22% in the presence of 20 microM sphingosine and phytosphingosine, respectively. This inhibition was completely abolished by deoxycholate at a concentration of 1.5 mM. These observations suggest that sphingosine may regulate activity of PLC delta1 in the cell.     

Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids

Mitochondrila DNA (mtDNA) is the only extrachromosomal DNA in humans. It is a small (16.5 kb) genome which encodes 13 essential peptides of the respiratory chain, two rRNAs and 22 tRNAs. Defects of this genome are now recognized as important causes of disease and may take the form of point mutations or rearrangements. There is no effective treatment for patients with mtDNA mutations. In the majority of patients with mtDNA defects, both mutant and wild-type molecules are present in the same cell-a phenomenon known as intracellular heteroplasmy. In addition, in the presence of heteroplasmy there is a threshold whereby a certain level of mutant mtDNA is necessary before the disease becomes biochemically and clinically apparent. Based on the presence of heteroplasmy and the recessive nature of these mutations, we believe it will be possible to treat patients by selectively inhibiting the replication of the mutant mtDNA, thereby allowing propagation of only the wild-type molecule. To confirm the validity of such an approach we synthesised peptide nucleic acids (PNAs) complementary to human mtDNA templates containing a deletion breakpoint or single base mutation, both mutations well documented to cause disease. Using an in vitro replication run-off assay under physiological conditions, the antigenomic PNAs specifically inhibited replication of mutant but not wild-type mtDNA templates. Furthermore, we have shown uptake of these PNAs into cultured human myoblasts. We believe that we have therefore established the potential value of antigenomic PNA therapy for patients with heteroplasmic mtDNA disorders.     

Effect of nucleic acid reactive antibodies on tumor cells grown in vivo

Nucleic acid reactive antibodies have been reported to inhibit various nucleic acid mediated functions in cell free systems. These antibodies were also shown to inhibit the growth of transformed cells in culture due to the high rate of endocytosis in transformed cells as compared to normal cells. In this report, we have tested the possibility of nucleic acid reactive antibodies inhibiting the growth of tumor cells in vivo. The life span of mice bearing Dalton's lymphoma ascites tumor cells was increased, when they were immunized with conjugates of guanosine-BSA, GMP-BSA and tRNA-MBSA complex before transplanting the tumor cells. A similar effect was also observed when mice were injected intraperitoneally with antibodies to guanosine or GMP along with the tumor cells. The specificity was ascertained, as immunization with non-specific antigens did not show any significant effect on tumor bearing mice. The results shows that nucleic acid reactive antibodies inhibit the growth of tumor cells in vivo.     

A toxic shock syndrome toxin-1 peptide that shows homology to amino acids 180-193 of mycobacterial heat shock protein 65 is presented as conventional antigen

Peripheral blood mononuclear cells (PBMC) from the majority of adults (12 out of 18 subjects tested) showed an in vitro proliferative response to a 20 amino acid long peptide (peptide 1, a.a 18-37) derived from TSST-1. In contrast, thymocytes and PBMC from cord blood did not proliferate to this peptide. TSST-1 peptide 1 did not induce IL-1 beta mRNA in monocytes indicating that it does not behave as a superantigen. Proliferation of PBMC to peptide 1 could be blocked by anti-HLA-DR, but not by anti-HLA DP or DQ monoclonal antibodies suggesting that HLA-DR molecules are the restriction elements for the recognition of this peptide by T cells. Studies with subjects of known HLA-DR types showed that all types tested are capable of responding to this peptide. Peptide 1 shows homology to a.a 180-193 of mycobacterial hsp 65 and was shown to stimulate the proliferation of T cell lines and T cell clone specific for the purified protein derivative (PPD) of Mycobacterium tuberculosis. This cross reactivity may confer on TSST-1 the potential to trigger self reactivity and may also contribute to the natural immunity against TSST-1.     

Regulation of E-cadherin-mediated adhesion in Langerhans cell-like dendritic cells by inflammatory mediators that mobilize Langerhans cells in vivo

Adhesion of Langerhans cells (LC) to keratinocytes is mediated by E-cadherin. IL-1, TNF-alpha, and LPS mobilize LC from epidermis and presumably attenuate LC-keratinocyte adhesion. To determine whether these mediators modulated LC E-cadherin-dependent adhesion directly, we characterized their effects on LC-like dendritic cells expanded from murine fetal skin (FSDDC). FSDDC were propagated from day 16 C57BL/6 fetal skin and isolated as aggregates (FSDDC-A) in which homophilic adhesion was mediated by E-cadherin. IL-1, TNF-alpha, and LPS induced dissociation of FSDDC-A that began within 4 to 8 h and was complete within 20 h. Anti-IL-1RI mAb inhibited disaggregation caused by IL-1alpha and IL-1beta, but not that induced by TNF-alpha or LPS. Anti-TNF-alpha mAb inhibited the effect of TNF-alpha and LPS, but not that caused by IL-1alpha or IL-1beta. Flow cytometry of FSDDC-A revealed that IL-1, TNF-alpha, and LPS induced increased expression of MHC class II, CD40, and CD86 and decreased E-cadherin expression that was temporally related to dissociation of aggregates. IL-1 and TNF-alpha caused a rapid reduction in FSDDC E-cadherin mRNA levels that preceded the decrease in E-cadherin surface expression. These results demonstrate that cytokines that induce LC emigration in vivo act directly on LC-like cells in vitro, reduce E-cadherin mRNA levels, down-regulate E-cadherin surface expression, and induce a loss of E-cadherin-mediated adhesion.     

Schistosoma mansoni: genetic complementation analysis shows that two independent hycanthone/oxamniquine-resistant strains are mutated in the same gene

Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide whether they are both mutated in the same gene with respect to drug-sensitive schistosomes. One of the two strains was isolated in the laboratory, while the other one originated from a treated uncured patient and was subsequently drug selected in the laboratory. The approach consisted in a genetic complementation test performed essentially by crossing the two strains and assessing resistance in the progeny. Since no reappearance of drug sensitivity was detected in the progeny, it was concluded that the two strains failed to complement and were therefore mutated in the same gene. This finding suggests that a single step of drug activation operates in sensitive schistosomes and is ineffective in resistant worms.     

Direct in vivo gene transfer and expression in malignant cells using adenovirus vectors

To evaluate the ability of replication-deficient, recombinant adenovirus vectors to transfer genes to human tumor cells in vivo, adenovirus vectors containing the Escherichia coli lacZ (Ad.RSV beta gal) gene (coding for beta-galactosidase; used as a cell marker for gene transfer) or the human alpha 1-antitrypsin (Ad-alpha 1AT) cDNA (used as an example of a secreted protein) were administered intraperitoneally to nude mice with human malignant mesothelioma cell (H-MESO-1) malignant ascites. Preliminary in vitro studies showed that both vectors effectively transferred genes to H-MESO-1 cells. Tumor cells recovered from ascites of animals intraperitoneally administered a control adenovirus revealed no evidence of beta-galactosidase (beta-gal) activity 3 or 14 days later. In contrast, beta-gal activity was detected at the same time points in tumor cells from animals receiving intraperitoneal Ad.RSV beta gal. Flow cytometric quantification of beta-gal activity in recovered cells showed < 3% beta-gal-positive cells in animals administered control virus, but in animals administered intraperitoneal Ad.RSV beta gal there was a mean of 71 +/- 18% positive cells at 3 days and 56 +/- 27% at 14 days. Human alpha 1AT was not detected by enzyme-linked immunosorbent assay (ELISA) in ascites of animals receiving a control virus; however, in ascites of animals administered Ad-alpha 1AT, 21,000 +/- 3,800 ng/ml of human alpha 1AT was detected at 3 days and 4,900 +/- 1,700 ng/ml at 14 days. These data demonstrate that replication-deficient recombinant adenovirus vectors can be used to transfer genes to malignant cells in vivo and suggest a new strategy for genetic modification for antitumor therapy.     

Specific immune induction following DNA-based immunization through in vivo transfection and activation of macrophages/antigen-presenting cells

The initiation of an adaptive immune response requires Ag presentation in combination with the appropriate activation signals. Classically, Ag presentation and immune activation occur in the lymph node and spleen, where a favorable organ architecture and rich cellular help can enhance the process. Recently, several investigators have reported the use of DNA expression cassettes to elicit cellular and humoral immunity against diverse pathogens. Although the immune mechanisms involved are still poorly understood, plasmid inoculation represents a model system for studying immune function in response to invading pathogens. In this report, we demonstrate the presence of activated macrophages or dendritic cells in the blood lymphocyte pool and peripheral tissues of animals inoculated with DNA expression cassettes. These cells are directly transfected in vivo, present Ag, and display the surface proteins CD80 and CD86. Our studies indicate that these cells function as APC and can activate naive T lymphocytes. They may represent an important first step APC in genetic immunization and natural infection.     

Regulation of alkaline phosphatase gene expression in human hepatoma cells by bile acids

HHV8 is a new herpesvirus recently identified in the Kaposi's sarcoma lesions, and initially named Kaposi's sarcoma-associated herpesvirus. It is a member of gamma-2 herpesvirus family and it shows a number of homologies with the Epstein-Barr virus and the herpesvirus saimiri. HHV8 genome also codes for several proteins which are homologous to cellular proteins and could disturb the regulation mechanisms of cellular proliferation and apoptosis. This is the case for a viral IL6, an antiapoptotic factor homologous to Bcl2, a viral cyclin, a member of the IRF family (interferon regulatory factors) and a G-protein-coupled receptor homologous to the IL8 receptor. Seroprevalence studies showed that HHV8 infection was not ubiquitous but rather limited to some geographic areas (Italy, Greece, Africa), and to some populations of homosexual and bisexual individuals with sexually transmitted diseases. To date, several lines of epidemiologic evidence suggest that this virus is sexually transmitted, although other routes of transmission cannot be excluded.     

Interferon-gamma-inducing factor gene transfection into Lewis lung carcinoma cells reduces tumorigenicity in vivo

The operative mortality and morbidity in patients with severe left ventricular dysfunction who undergo coronary artery bypass grafting (CABG) remain high. The low ejection fraction is the major risk factor for operative mortality. However, ejection fraction (EF) alone may not necessarily be an accurate predictor of operative mortality. We studied the correlation between indices of left ventricular volume and operative mortality. One thousand patients undergoing isolated coronary bypass operations were divided into three groups according to their preoperative ejection fraction. Fifty patients (group I) had severe left ventricular dysfunction (EF < or = 0.3), 56 patients (group II) had moderately left ventricular dysfunction (0.3 < EF < or = 0.4) and 894 patients (group III) had good left ventricular function (EF > 0.4). We analyzed the relationship between hospital mortality and left ventricular volume in 106 patients with an EF < or = 0.4. RESULTS: Cardiac index was not significantly different among the three groups. The left ventricular end-diastolic pressure (LVEDP) and mean pulmonary artery pressure in groups I an II were higher than those in group III. The left ventricular end-diastolic volume (LVEDV) was 146 +/- 44 ml/m2 in Group I, 112 +/- 31 ml/m2 in Group II and 82 + 30 ml/m2 in Group III, respectively (Group I versus II, p < 0.05, Group I and II versus III, p < 0.01). The left ventricular end-systolic volume (LVESV) was 111 +/- 38 ml/m2 in Group I, 72 +/- 21 ml/m2 in Group II and 30 +/- 14 ml/m2 in Group III, respectively (Group I versus II, p < 0.05, Group I and II versus III, p < 0.01). The LVEDV and LVESV were higher in Group I than in Group II and both in Groups I and II were higher than in Group III. The hospital mortality of any cause before discharge was 8.0% (4/50) in Group I, 3.6% (2/56) in Group II, and 2.0% (18/894) in Group III. The mortality in Group I was higher than that in Group III, but the mortality between Groups I and II was not different. We assessed correlations between large left ventricle with left ventricular dysfunction and operative mortality in 106 patients with ejection fractions of < or = 0.4. The hospital mortality in patients with both under fraction 0.4 and an LVESV > or = 140 ml/m2 was 50% (4/8). This rate was higher than in patients with an LVESV between 80 and 140 ml/m2 (1.8%, 1/55) (p = 0.0006) and an LVESV less than 80 ml/m2 (2.3%, 1/43), (p = 0.0013). The hospital mortality in patients with an LVEDV > or = 200 ml/m2 was 67% (4/6). It was also higher than that in patients with an LVEDV between 200 and 120 ml/m2 (1.7%, 1/58), (p = 0.0001), and an LVEDV less than 120 ml/m2 (2.4%, 1/42), (p = 0.0004). We conclude that patients with a low ejection fraction and an elevated LVESV or LVEDV are at increased risk for hospital death following CABG.     

Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.     

Molecular lysis of synovial lining cells by in vivo herpes simplex virus-thymidine kinase gene transfer

Herpes simples virus thymidine kinase (HSV-TK) expression plasmid DNA was injected into the joint space of rabbits with antigen-induced arthritis (AIA). Purified plasmid DNA was able to mediate transfection of synovial lining cells and transient overexpression of HSV-TK in the context of active synovial inflammation. The pharmacodynamic distribution of intraarticular expression plasmid DNA was confined to the joint space. Arthritic rabbits treated with intraarticular expression plasmid DNA followed by intravenous ganciclovir (GCV, 5 mg/kg) twice daily for 3 days showed histologic evidence of synovial lining layer cytolysis when articular tissues were examined 21 days posttreatment. There was also a reduction in joint swelling in the TK-treated knees. No untoward clinical effects were observed in the rabbits and no evidence of cytolytic damage specific to the TK-GCV gene therapy was observed either in the articular cartilage or bone. The application of TK-GCV intraarticular gene therapy using purified expression plasmid DNA for the induction of synovial cytolysis may be applicable to the treatment of human inflammatory arthritis.