In VivoStudies of Adenovirus-Based p53 Gene Therapy for Ovarian Cancer

Objectives.To test the safety, efficacy, and toxicity of gene therapy using wild-type p53-expressing adenovirus (Ad-CMV-p53) in a nude mouse model with intraperitoneal (ip) 2774 human ovarian cancer cell line that contains a p53 mutation.Study design.An initial study of adenovirus tolerance was determined in nude mice by a single ip injection of increasing doses of Ad-CMV-p53. Nude mice were implanted with an LD₁₀₀dose of 1 × 10⁷cells. To study the efficacy and specificity of Ad-CMV-p53 treatment, the mice received treatment with different adenovirus constructs. One group received Ad-CMV-p53 and another group received a control adenovirus construct, Ad-CMV-βgal. To study the treatment response to Ad-CMV-p53, the mice were divided into groups and received various treatment schedules of 1 × 10⁸pfu of Ad-CMV-p53.Results.The mice tolerated Ad-CMV-p53 without adverse effects at doses of 1 × 10⁸pfu. The response to Ad-CMV-p53 showed significant survival duration in each dose regimen, with a survival time greater than that of untreated animals (P= 0.0173). However, no statistically significant survival advantage was observed between Ad-CMV-p53- and Ad-CMV-βgal-treated mice.Conclusions.These studies show that at the adenovirus dose and administration regimen used, there is effective but not specific 2774 tumor growth inhibitionin vivo.Efficient introduction of biologically active genes into tumor cells would greatly facilitate cancer therapy. Thus, although promising, these results caution that much effort will be required to realize the potential for clinical application of adenovirus-based ovarian cancer gene therapy.     

The presence of messenger RNA for HLA class I in human platelets and its capability for protein biosynthesis

Summary. In order to determine whether platelets contain specific messenger RNA encoding for HLA class I molecules, polymerase chain reaction (PCR) was performed with RNA from different platelet donors. Two amplified 300 bp and 279 bp cDNA fragments were obtained which encompassed sequences from 321 to 620 and from 795 to 1073. The 300 bp fragment encodes exon 2 and exon 3, the 279 bp encodes a portion of exon 4, exon 5, exon 6 and a portion of exon 7. A 300 bp nested PCR product from one donor, that encoded for the highly polymorphic region α2, was cloned and sequenced. The resulting nucleotide sequences fitted to the expected sequence for HLA B*3801 of this donor. Sequence analysis of the 279 bp PCR product demonstrated the presence of exon 5 encoding for the 117 bp transmembrane domain.
In addition, de novo protein biosynthesis was studied by radioimmunoprecipitation of HLA class I molecules from ³⁵S-methionine metabolically labelled platelet lysates with a monoclonal antibody (mab) w6/32 specific for a monomorphic epitope on the heavy chain of HLA class I antigens. Analysis of the immunoprecipitates on SDS polyacrylamide gel electrophoresis showed a specific band with apparent molecular weight (M_r) of 44 kD corresponding to integral membrane HLA protein.
On the basis of these results, we conclude that platelets contain specific messenger RNA encoding for HLA class I molecules and have the capability to synthesize the integral HLA membrane protein.     

Myocardial perfusion imaging in humans by contrast echocardiography using polygelin colloid solution

This study evaluated the myocardial contrast effect and safety of polygelin colloid solution selectively injected into the coronary arteries in 25 patients during two-dimensional echocardiography. Six patients (group I) had selective intracoronary injections of nonagitated and 19 (group II) of hand-agitated polygelin colloid solution. Myocardial contrast was seen on two-dimensional echocardiographic cross sections in three patients of group I and in all patients of group II; in 16 patients it was also seen on M-mode echocardiograms. The contrast effect lasted for 15 to 60 seconds. The intensity of myocardial opacification was not significantly influenced by the amount of polygelin colloid solution injected, heart rate or cardiac size. The total number of contrast-enhanced segments after right and left coronary artery injections delineated the entire cross-sectional area in any given view. None of the patients developed symptoms during or immediately after the injections. One patient had transient second degree atrioventricular block after a right coronary wedge injection, one patient showed a QRS axis shift and two others had transient T wave changes. There were no aortic blood pressure changes and no significant serum enzyme (creatine kinase [CK], CK-MB fraction, glutamic oxaloacetic transaminase) elevation or alterations of left ventricular function assessed echocardiographically. It is concluded that hand-agitated polygelin colloid solution is a useful and safe intracoronary contrast agent for delineating myocardial perfusion areas on two-dimensional echocardiography in humans.     

Autoantibody-mediated complement activation on platelets is a common finding in patients with immune thrombocytopenic purpura (ITP)

Background: It is commonly accepted that antibody‐mediated removal of platelets represents a major mechanism of platelet destruction in immune thrombocytopenic purpura (ITP). Although complement activation may participate in platelet clearance, frequency and specificity of complement activation have not yet been studied systematically in ITP. Patients and methods: We examined blood samples from 240 patients with ITP. Samples were assessed for the presence of free and bound platelet autoantibodies by a standard glycoprotein‐specific assay (monoclonal antibody‐specific immobilization of platelet antigens). The ability of all sera to fix complement to a panel of human platelets was investigated in a complement fixation (CF) assay. Fixation of C1q to isolated GP IIb/IIIa was assessed by flow cytometry. Results: Glycoprotein‐specific autoantibodies were detected as platelet‐bound antibodies in 129 (54%) and as additional free antibodies in 26 (11%) and were undetectable in 111 (46%) patients. Assessing these subgroups for CF, 103 (65%), 21 (81%), and 33 (30%) sera gave positive results. If GP IIb/IIIa was absent from the test platelets, 81 (67%) lost their ability to fix complement; if GP Ib/IX was absent, 37 (30%) lost their ability to fix complement. C1q fixation to immunobeads coated with GP IIb/IIIa was observed in 50% of sera containing anti‐GP IIb/IIIa antibodies. Conclusions: In a significant number of patients with chronic ITP, platelet autoantibodies are capable of activating the classical complement pathway. CF is even present in ITP sera without detectable autoantibodies, indicating that current techniques for autoantibody detection may be insufficient. The major targets for complement‐fixing autoantibodies in ITP are GP IIb/IIIa and GP Ib/IX.     

Nucleoside triphosphates inhibit ADP,collagen, and epinephrine-induced platelet aggregation: Role of P2Y1 and P2Y12 receptors

Background

Platelets express two ADP receptors namely P2Y₁ and P2Y₁₂ that regulate ADP and other agonists-induced platelet aggregation. P2Y₁ receptor activation causes platelet shape change while P2Y₁₂ receptor activation induces platelet aggregation. Previously, anti-aggregatory effects of ATP on ADP-induced and pro-aggregatory effects on epinephrine-induced platelet aggregation have been reported. However, the effects of other nucleoside triphosphates on platelet aggregation have never been described. The aim of the present study was to characterise the effects of nucleoside triphosphates (ATP, UTP, GTP, and CTP) on agonist-induced platelet aggregation.

Methods

The experiments were performed on platelet rich plasma freshly isolated from blood donated by healthy human volunteers.

Results

All the nucleoside triphosphates tested inhibited ADP- and collagen-induced platelet aggregation in a concentration-dependent manner with a rank order of potency, 2MeSATP > ATP ≥ α,β,methyleneATP > UTP  >> CTP ≥ GTP. The IC₅₀ values against ADP (10 μM)-induced platelet aggregation were 0.039 ± 0.013, 18 ± 7, 25 ± 6, 32 ± 9, 360 ± 130, and 400 ± 160 μM, respectively. Low concentrations of ATP induced platelet shape change which was due to contaminating ADP. However, higher concentrations antagonised ADP and MRS2365-induced platelet shape change. The ATP analogue α,β,methyleneATP and CTP but not UTP and GTP also antagonised ADP-induced platelet shape change. Similarly, low ATP concentrations potentiated epinephrine-induced platelet aggregation that was abolished by P2Y₁ antagonist MRS2500 suggesting P2Y₁ receptor activation due to contaminating ADP. Higher ATP concentrations, α,β,methyleneATP, UTP, CTP, and GTP antagonised epinephrine-induced platelet aggregation.

Conclusion

Thus, the data demonstrate nucleoside triphosphates in general act as P2Y₁₂ receptor antagonists and antagonise ADP-, collagen-, and epinephrine-induced platelet aggregation.