Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study.

We have established a number of 293T cell lines that express a human anti HIV-1 factor APOBEC3G. Out of seven cell clones examined, four were readily demonstrated to express APOBEC3G by immunoblotting analysis. In particular, two clones (A3G-C1 and -C4) were found to produce a much higher level of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all these cell clones were similar to that of the parental cells, producing a comparable level of virions upon transfection of wild type and vif-minus proviral DNA clones. Furthermore, the expression level of APOBEC3G in the best cell line (A3G-C1) was far much higher than those of an APOBEC3G-positive lymphocyte cell line and peripheral blood mononuclear cells. We finally monitored the incorporation of APOBEC3G into virions produced in A3G-C1. APOBEC3G was easily detected in progeny viral particles upon transfection of vif-minus proviral clone but not of wild type. These results indicated that our new A3G-C1 cell line is eminently useful for various studies on the interaction of human APOBEC3G and HIV-1 Vif.

Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions.

A host cytidine deaminase, APOBEC3G (A3G), inhibits replication of human immunodeficiency virus type 1 (HIV-1) by incorporating into virions in the absence of the virally encoded Vif protein (Deltavif virions), at least in part by causing G-to-A hypermutation. To gain insight into the antiretroviral function of A3G, we determined the quantities of A3G molecules that are incorporated in Deltavif virions. We combined three experimental approaches-reversed-phase high-pressure liquid chromatography (HPLC), scintillation proximity assay (SPA), and quantitative immunoblotting-to determine the molar ratio of A3G to HIV-1 capsid protein in Deltavif virions. Our studies revealed that the amount of the A3G incorporated into Deltavif virions was proportional to the level of its expression in the viral producing cells, and the ratio of the A3G to Gag in the Deltavif virions produced from activated human peripheral blood mononuclear cells (PBMC) was approximately 1:439. Based on previous estimates of the stoichiometry of HIV-1 Gag in virions (1400-5000), we conclude that approximately 7 (+/-4) molecules of A3G are incorporated into Deltavif virions produced from human PBMCs. These results indicate that virion incorporation of only a few molecules of A3G is sufficient to inhibit HIV-1 replication.

Quantitation of cytosine deaminase mRNA by real-time reverse transcription polymerase chain reaction: a sensitive method for assessing 5-fluorocytosine toxicity in vitro.

Cytosine deaminase/5-fluorocytosine (CD/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), semiquantitative immunocytochemistry (ICC), and 5-[(3)H]FC enzyme assay, respectively. Results with human colon (LS174T) cancer cells infected with a replication-incompetent adenovirus encoding CD (AdCMVCD) demonstrated a significant correlation between CD mRNA and enzyme activity up to 24 h postinfection. A direct correlation was found between CD dose (AdCMVCD PFU/cell) and CD mRNA and protein expression (P < 0.002) in both LS174T and BxPC-3 pancreatic cancer cells, but the relationship with enzyme activity was less strong in LS174T cells (P = 0.09). A remarkable concordance existed among Q-RT-PCR, ICC and enzyme assays with both cell lines. Importantly, CD dose and mRNA and protein expression inversely correlated with 5-FC IC(50) (P < 0.02). Quantitation of CD markers also facilitated identification of factors governing differential susceptibility to CD/5-FC. These results suggest that Q-RT-PCR will be useful for monitoring transgene expression in future studies using improved CD-based expression vectors and may also be useful in predicting the response to CD/5-FC therapy, which is likely to be heterogeneous in the patient population.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the 6.7 K/gp19 K region.

The use of genetically engineered, replication-selective viruses to treat cancer is being realized with viruses such as ONYX-015, a human adenovirus that selectively destroys p53 mutant cancer cells. To enhance further the clinical efficacy of ONYX-015 and viruses like it, we have developed a novel gene delivery system for replicating adenoviruses. This system has two unique features. First, it uses the endogenous adenoviral gene expression machinery (promoter, splicing, polyadenylation) to drive transgene expression. Second, a single region or gene in the multi-gene E3 transcription unit is selectively substituted for by the therapeutic transgene(s). Analyzing various transgene substitutions for the 6.7 K/gp19 K region of E3, we demonstrate the following: (1) transgene expression in this system is predictable and mimics the substituted endogenous gene expression pattern, (2) expression of surrounding E3 genes can be retained, (3) the insertion site choice can effect both the transgene expression level and the viral life cycle, and, (4) expression levels from this system are superior to those generated from a replication-defective virus using the HCMV enhancer-promoter and this is dependent on viral DNA replication. This unique methodology has broad application to the rapidly evolving field of replicating virus-based therapies.

Detection of cytosine deaminase in genetically modified tumor cells by specific antibodies.

Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy.

The measurement of nucleoside deaminases by high performance liquid chromatography and their use in clinical chemistry.

The measurement of the nucleoside deaminases--cytidine deaminase, guanosine deaminase and adenosine deaminase--by reversed phase high performance liquid chromatography is reviewed. The clinical value of assaying the enzyme activity is discussed for each of these enzymes. Both cytidine deaminase and adenosine deaminase measurements have proven clinical value, although the use of the assay of cytidine deaminase in the diagnosis of pre-eclampsia is probably not helpful.

Value of adenosine deaminase estimation in the diagnosis of tuberculous ascites.

Adenosine deaminase was estimated in ascitic fluids of 49 patients with ascites (19 tuberculous, 20 cirrhotic, and 10 malignant). The adenosine deaminase concentration in tuberculous ascitic fluid was 98.8 +/- 20.1 U/L (mean +/- SD), which was significantly more than that noted in cirrhotic (14 +/- 10.6 U/L) or malignant (14.6 +/- 6.7 U/L) ascitic fluids (p less than 0.001 for each). At a cut-off value of greater than 33 U/L, the sensitivity, specificity, positive and negative predictive value, and the overall diagnostic accuracy for diagnosing tuberculous ascites were 100%, 96.6%, 95%, 100%, and 98%, respectively. We conclude that estimation of adenosine deaminase in ascitic fluid is an easy and reliable method for diagnosing tuberculous ascites.

[Gamma interferon and adenosine deaminase in pleuritis]. / Interferón gamma y adenosina desaminasa en las pleuritis.

The concentration of gamma interferon (IFN gamma) and adenosine deaminase (ADA) activity were measured in the pleural fluid of 162 patients to compare their diagnostic significance and to establish a possible correlation between both tests. The IFN gamma levels in tuberculous pleural effusions were quite variable, with a mean of 93 U/ml and a median of 48 U/ml. They were higher than 2 U/ml in all cases, whereas no case of nontuberculous effusion showed higher values. ADA activity was higher than 43 U/l in all tuberculous effusions, with a mean value of 80 U/l, higher than in any other group except lymphoma. In three pleural effusions associated with lymphoma, one with mesothelioma, one with adenocarcinoma and four with empyema, ADA activity was higher than 43 U/l. In these patients, IFN gamma levels were low. There was no correlation in the whole series or in any group between IFN and ADA. Both parameters can be very useful for the diagnosis of tuberculous pleuritis. The measurement of IFN gamma is more specific, although the experience with this test is more limited than with ADA. It has the additional shortcoming of a high cost and it requires facilities for radionuclide use. Therefore, we think that ADA measurement should be considered as a routine test while IFN gamma measurement should be reserved for reference institutions.

Adenosine deaminase complexing protein (ADCP) expression and metastatic potential in prostatic adenocarcinomas.

The expression of the adenosine deaminase complexing protein (ADCP) was investigated by immunohistochemistry in the normal and hyperplastic human prostate, in 30 prostatic adenocarcinomas, and in seven human prostatic adenocarcinoma cell lines grown as xenografts in athymic nude mice. In the normal and hyperplastic prostate, ADCP was localized exclusively in the apical membrane and the apical cytoplasm of the glandular epithelial cells. In prostatic adenocarcinomas, four distinct ADCP expression patterns were observed: diffuse cytoplasmic, membranous, both cytoplasmic and membranous, and no ADCP expression. The expression patterns were compared with the presence of metastases. We found an inverse correlation between membranous ADCP immunoreactivity and metastatic propensity. Exclusively membranous ADCP immunoreactivity occurred only in non-metastatic tumours. In contrast, the metastatic tumours showed no or diffuse cytoplasmic ADCP immunoreactivity. This suggests that immunohistochemical detection of ADCP might predict the biological behaviour of prostatic cancer. However, the occurrence of membranous ADCP immunoreactivity in the xenograft of a cell line (PC-EW), derived from a prostatic carcinoma metastasis, indicates that not only the tendency to metastasize modulates ADCP expression.

The protonated form of 1-N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine is identified at the active site of adenosine deaminase.

A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), 1-N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine (epsilon-EHNA), is protonated at the active site of the enzyme. In epsilon-EHNA [K1 = (4.06 +/- 1.00) 10(-6) M] part of the competitive inhibition of EHNA is combined with spectroscopic properties of etheno-adenines. Computer subtraction of the fluorescence excitation spectrum of ADA from that of its equimolar complex with epsilon-EHNA yielded the corrected excitation spectrum of epsilon-EHNA at the active site of the enzyme. This spectrum mimics that of epsilon-EHNA at pH 5.5 in buffer solution and is suggested to indicate a shift in protonation equilibrium at the active site.

Correlation between tumor histology, steroid receptor status, and adenosine deaminase complexing protein immunoreactivity in ovarian cancer.

Adenosine deaminase complexing protein (ADCP) immunoreactivity was investigated in 40 ovarian tumors and correlated with clinicopathologic parameters, including tumor steroid receptor content. Ten (29%) of 34 common epithelial ovarian carcinomas showed ADCP reactivity. Reactivity for ADCP was seen more frequently in mucinous (100%; p less than 0.001), well-differentiated (73%; p less than 0.001) and Stage I (56%; p less than 0.05) ovarian carcinomas. Furthermore, tumors that contained high levels of androgen receptors and tumors that did not contain estrogen receptors were more frequently ADCP positive (p less than 0.05). However, after stratifying for histologic grade, no correlation between ADCP reactivity and receptor status was found. Determination of ADCP reactivity appears to be of limited value in ovarian cancer.

Increased erythrocyte adenosine deaminase activity in children with perinatal human immunodeficiency virus infection.

Erythrocyte adenosine deaminase (ADA) activity was assessed in 33 children born to human immuno-deficiency virus (HIV)-positive mothers. The enzyme values were significantly increased in infected, symptom-free children compared with a control group of HIV-negative subjects (mean +/- SD: 0.34 +/- 0.01 unit/ml red blood cells (RBC) vs. 0.25 +/- 0.04 unit/ml RBC, P less than 0.01) and a further significant increase was found in symptomatic children (0.45 +/- 0.02 unit/ml RBC, P less than 0.01 vs. infected, symptom-free children). ADA values were slightly enhanced also in the group of infants in whom the state of HIV infection was indeterminate (0.29 +/- 0.03 unit/ml RBC, P not significant vs. controls). These data indicate that increased erythrocyte ADA activity may be a useful though indirect marker of HIV infection in children at risk and be of possible prognostic relevance. Since increased values were present also in children without overt infections or hematologic disorders, and ADA activity of erythrocytes obtained from healthy donors did not increase after 1 hour incubation with patients' serum, HIV could induce large amounts of cellular enzyme infecting directly erythroid precursor cells.

Diagnostic value of adenosine deaminase and gamma-interferon in tuberculous and malignant pleural effusions.

It has been found that both adenosine deaminase (ADA) and gamma-interferon (r-IFN) are higher in tuberculous pleural effusions than in other types of pleural effusion. Both mechanisms may be related to the T-lymphocytes in effusions. We studied 39 pleural effusions: 19 tuberculous and 20 malignant [The ADA was determined according to the Giusti method and r-IFN by ELISA]. We compared the ADA and r-IFN levels in both groups of effusions and tried to determine if there was any correlation between them. The results showed: (1) The ADA enzyme level was significantly higher in tuberculous effusions than in malignant effusions as was expected (133.0 +/- 50.4 vs 32.0 +/- 17.7 U/L). (2) The r-IFN was also significantly higher in tuberculous effusions than in malignant effusions (30.4 +/- 17.4 vs 2.9 +/- 2.8 U/ml). (3) The coefficients of regression for the ADA and r-IFN levels were poor. In conclusion, both tests for measuring ADA and r-IFN levels are excellent methods for differentiating tuberculous and malignant effusions, and especially measurement of the r-IFN level could serve as a more specific test for differentiating malignant pleural effusions with high ADA levels. However, no strong correlation was found between the ADA and r-IFN levels.

Guillain-Barré syndrome and tularemia pleuritis with high adenosine deaminase activity in pleural fluid.

A 56-year-old man suffered from prolonged fever, sore throat and cough, followed by pleural effusion and reversible progressive ascending muscle weakness. The condition fulfilled the diagnostic criteria of Guillain-Barré syndrome. Tuberculosis was initially suspected because of lymphocyte predominance and high adenosine deaminase activity in the pleural fluid. Later, an agglutination titer of 10,240 to Francisella tularensis antigen was found and an infected hare exposure could be identified. Thus, the activity of adenosine deaminase may be high also in tularemia pleuritis.

Diagnostic value of ascites adenosine deaminase in tuberculous peritonitis.

The value of ascitic fluid adenosine deaminase activity in distinguishing tuberculosis from other causes of ascites was examined in a retrospective study of 41 patients with bacteriologically confirmed tuberculous peritonitis and 41 control patients, matched for age and sex, with ascites of other causes (12 alcoholic cirrhosis, 5 cryptogenic cirrhosis, 12 malignant disorders, 3 pancreatitis, and 9 miscellaneous causes). The mean ascites adenosine deaminase activity was 99.8 (SD 49.1) in tuberculous patients and 14.8 (8.4) U/l in control patients (p less than 0.0001). A cutoff of 32.3 U/l had a sensitivity of 95% and specificity of 98% in distinguishing between the two groups. In a subsequent prospective study of 64 patients with ascites, 11 were found to have tuberculosis. Of the others, 23 had cirrhosis (18 alcoholic, 5 cryptogenic), 17 malignant disorders, 3 pancreatitis, 5 cor pulmonale, 3 congestive cardiac failure, 1 systemic mastocytosis, and 1 renal failure and hypothyroidism. The mean ascites adenosine deaminase activity was 112.6 (45.0) U/l in the patients with tuberculous ascites and 16.3 (36.7) U/l (p less than 0.0001) in those with ascites of other causes. In this study, adenosine deaminase had a sensitivity of 100% and specificity of 96% in discriminating tuberculosis from other causes of ascites. These findings suggest that the ascitic fluid adenosine deaminase activity may be used to identify patients in whom the diagnosis of abdominal tuberculosis must be pursued.