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J L Casey

Publications and source records attributed to J L Casey.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cloning and characterization of partial cDNAs for woodchuck cytokines and CD3epsilon with applications for the detection of RNA expression in tissues by RT-PCR assay.

Immunologic reagents and methodology are essential to develop further the woodchuck and woodchuck hepatitis virus (WHV) as a model of immune response, inflammation, and immunotherapy in hepatitis B virus (HBV) infection. Partial cDNA clones for the woodchuck CD3epsilon marker of T cells (536 bp) and for selected woodchuck cytokines were developed, including IL-1beta (332 bp), IL-2 (249 bp), IL-4 (205 bp), IL-10 (476 bp), IFN-gamma (476 bp), and TNF-alpha (381 bp). This panel of markers includes sets to measure RNAs for T cells (CD3epsilon), immune response induction (IL-1beta, IL-2), TH subsets (TH1, IL-2/IFN-gamma vs. TH2, IL-4/IL-10), and effector molecules that regulate hepadnavirus replication and liver injury (IFN-gamma, TNF-alpha). Primers representing highly conserved segments of genes from other species were used to derive the partial cDNA clones. Target RNA was obtained from woodchuck peripheral blood mononuclear cells (PBMC) that were stimulated in vitro with ConA, LPS, and human rIL-2. The cDNA clones were validated by 1) comparison with other species for homologies in the nucleotide and predicted amino acid sequences and 2) a first generation assay demonstrating induction of the respective RT-PCR products in stimulated woodchuck PBMC. The corresponding RNAs were also detectable in most cases in the total RNA from the livers of uninfected and WHV-infected woodchucks and differential expression of IFN-gamma and TNF-alpha RNAs was suggested. Second generation, semi-quantitative assays for the RNAs were validated using RT-PCR and dot-blot hybridization with 32P-oligomers derived from the internal sequences of the respective clones. Continued study of the woodchuck immune response to WHV infection using these assays will provide insight into the kinetics and immune mechanisms that initiate and maintain chronic hepadnavirus infection and, hence, enable development of improved immunotherapies for established chronic HBV infection.

Amino Acid Sequence↗
PubMed1997

The predominance of hepatitis delta virus genotype I among chronically infected Italian patients.

The sera of 46 Italian patients with chronic hepatitis delta virus (HDV) infection were analyzed for HDV RNA by polymerase chain reaction (PCR) amplification. Genetic analysis of sequences amplified from two regions of the HDV genome indicated that all HDV RNA-positive patients (98%) were infected with HDV genotype I. In Italy, infection with this genotype appeared to be associated with a broad spectrum of chronic disease. No subtypes of HDV genotype I were identified, nor were genetic variations clearly associated with different disease patterns; however, clustering of some sequences suggested correlations with geography and transmission route. Italian HDV genotype I sequences were more diverse than those from east Asia and North America, suggesting that HDV genotype I was likely introduced to Italy earlier and/or from multiple sources as compared to those areas. All sequences analyzed were predicted to fold into the unbranched rod structure typical of HDV RNA. Within this structure, three conserved features were identified, including sequences around the RNA editing site and the polyadenylation signal site. We conclude that in Italy, where HDV infection has been endemic, the overwhelmingly predominant genotype of HDV is genotype I.

Adult↗
PubMed1996

RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase.

Hepatitis delta virus (HDV) is a subviral human pathogen that requires hepatitis B virus (HBV) for packaging. Concurrent infection by HBV and HDV increases the risk of severe liver disease compared to infection with HBV alone. The HDV genome is a closed circular RNA of about 1,700 bases which is replicated through an RNA intermediate, the antigenome. Both RNAs can be folded into highly base-paired, rod-shaped structures, similar to the plant viroid RNAs. Two forms of the sole HDV protein, hepatitis delta antigen, are derived from a single open reading frame by RNA editing; the enzymes responsible for the editing have not been characterized. Here we report that the purified enzyme dsRAD (for double-stranded-RNA-adenosine deaminase) can edit HDV antigenomic RNA in vitro. Most important, we observe that mutations in critical sequences of the antigenome have identical effects on in vitro and in vivo editing, suggesting that dsRAD, or a closely related enzyme, is responsible for editing HDV RNA in vivo.

Adenosine↗
PubMed1996

Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab' fragments.

The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab' fragments in nude mice bearing human colorectal tumour xenografts. The cross-linkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy beta-emitter. We have also compared the above antibody forms conjugated to both 131I and 90Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37 degrees C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab')2 and DFM and elevated splenic uptake of DFM labelled with 90Y was observed. Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 10(5) Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab')2 radiolabelled with 131I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 131I and 90Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for 131I-labelled antibodies. Because of the faster clearance, it should be possible to administer a higher total dose of 90Y-labelled TFM than IgG, which is attractive for RIT. Both A5B7 DFM and TFM, therefore, show favourable properties compared with their parent antibody forms.

Animals↗
PubMed1996

Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library.

We present a system for cancer targeting based on single-chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag. Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage. We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)-producing cancers using an iodine-123 labeled scFv anti-CEA selected for high affinity. All known tumor deposits were located, and advantages over current imaging technology are illustrated. ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

Adult↗
PubMed1996

Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F.

Recurring outbreaks of acute hepatitis have been a significant cause of morbidity and mortality among Peruvian military personnel stationed in the Amazon Basin region of Peru. The role of hepatitis B virus (HBV) and hepatitis D virus (HDV) infection was investigated as the possible cause of acute hepatitis among 88 military patients stationed at four different jungle outposts during 1992-1993. Analysis of serum markers indicated that 95% (84/88) had evidence of acute HBV infection; 64% (54/84) were also infected with HDV. Genetic analysis of PCR-amplified HDV and HBV fragments showed exclusively HDV genotype III and HBV genotype F. Furthermore, HDV RNA sequences were similar among patients from the same outpost but different from those at other jungle locations. The data suggested focal sources of HDV infection in the jungle environment of the outposts and, further, confirmed the unique association of HDV genotype III with severe cases of human disease in northern South America.

Acute Disease↗
PubMed1996

Hepatitis delta virus. Genetics and pathogenesis.

After the discovery of HDV there have been significant advances in the understanding of the biology and disease of HDV infection. Analyses at the molecular level have revealed several fascinating features (ribozyme activity, RNA-dependent RNA polymerase activity of RNA polymerase II, HDAg isoprenylation, and RNA editing) that are of significant interest. Intensive investigation of the ribozyme elements has yielded important insights in both functional and structural features. However, there is information lacking about other aspects of the HDV replication cycle including the specific nature of the interaction between HDAg and HDV RNA, the function of HDAg in HDV RNA replication, transcription by RNA polymerase II, and the mechanisms of HDV RNA editing and its regulation. Further study of these and other aspects of the HDV replication cycle will continue to enrich our understanding of basic biology. Evaluation of the mechanisms of HDV disease remains an important goal in the study of this agent. Although both acute and chronic disease are commonly associated with unfavorable outcomes, it is clear that chronic infection is associated with a broad spectrum of disease. The interactions between HDV, HBV, and the host are necessarily complex, and it is likely that each contribute factors that influence disease and outcome. Recent analyses of HDV genotypes have suggested that disease variations may be associated with viral genetic factors. Consistent with the obligate role of HBV in the HDV life cycle, HBV replication is also an important determinant of HDV disease. It is still unclear if interactions between specific genotypes or variants of HBV and HDV influence disease. Recent data also suggest that infection with multiple hepatitis viruses (HBV, HDV, and HCV) can influence the severity of disease. It remains to be seen whether coinfection with the recently discovered hepatitis G virus is associated with altered disease patterns. Further advances in our understanding HDV disease and possible therapeutic approaches will rely on a combination of additional studies at the molecular, genetic, epidemiologic, and clinical levels. These studies will continue to elaborate the model of HDV infection and disease that can ultimately be tested by experimental infection of chimpanzees and woodchucks.

Animals↗
PubMed1996

Technetium-99m radiolabeling using a phage-derived single-chain Fv with a C-terminal cysteine.

UNLABELLED: Single-chain Fv (scFv) antibody fragments have potential for clinical imaging because of their rapid tumor penetration and high tumor-to-tissue ratios at early time points. ScFvs clear rapidly from the circulation so radiolabels such as 99mTc which have short half-lives are desirable, but the free thiol groups necessary for labeling with 99mTc are not normally found on these molecules. METHODS: We constructed a vector which enabled a free cysteine to be linked to the C-terminus of scFvs. MFE-23, a scFv directed against carcinoembryonic antigen (CEA), was cloned into this vector and cys-tagged MFE-23 was labeled with 99mTc using a D-glucarate transfer method. RESULTS: The radiolabeled product was stable in vivo and in vitro and showed favorable tumor-to-blood ratios in vivo at early time points (4:1 at 24 hr and 8:1 at 48 hr), although high kidney levels were also detected. CONCLUSION: Our study demonstrates an effective method to enable scFvs radiolabeling with 99mTc and also shows the potential of using a 99mTc-labeled scFv for clinical imaging studies.

Adenocarcinoma↗
PubMed1995

Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography.

A new procedure is described for the purification of an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, from bacterial supernatant. A simple insertion of a hexa-histidine tail fused at the C-terminus (MFE-23 His) provides an affinity tag which selectively binds to transition metal ions immobilised on an iminodiacetic acid (IDA) derivitised solid phase matrix. This method proved to be superior to standard CEA antigen affinity chromatography in the following ways. (1) A higher yield was produced (10 mg/l as opposed to 2.2 mg/l of bacterial supernatant). The latter figure was largely affected by the limited availability (size of the column) of immobilised CEA antigen. (2) Scale-up was relatively simple and less costly. (3) The risk of tumour derived antigen leaching from the column is eliminated. Results showed that immobilised Cu2+ ions were more effective than Ni2+ and Zn2+ ions in retaining the His tagged product giving a 90% pure product on elution. Clinical grade material was generated using size exclusion chromatography to remove aggregated material, and Detoxi gel to remove bacterial endotoxins. Validation assays to measure DNA, copper and endotoxins were performed to assess the levels of contaminants. MFE-23 His retained 84% antigen binding after 6 months storage at 4 degrees C and > 75% after radiolabelling. Further experiments confirmed that the His tail did not affect biodistribution and tumour localisation in nude mice bearing human colorectal tumour xenografts.

Amino Acid Sequence↗
PubMed1995

Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA.

RNA editing plays a central role in the life cycle of hepatitis D virus (HDV), a subviral human pathogen. Previous studies (J.L. Casey, K.F. Bergmann, T.L. Brown, and J.L. Gerin, Proc. Natl. Acad. Sci USA 89:7149-7153, 1992; H. Zheng, T.-B. Fu, D. Lazinski, and J. Taylor, J. Virol. 66:4693-4697, 1992) had concluded that the genomic RNA of HDV was the target for RNA editing and that the editing reaction was a conversion of U to C. However, we show here that the antigenomic RNA of HDV is in fact the target for HDV RNA editing, which is therefore a conversion of A to G. This result is verified by using an assay specific for editing on the antigenomic RNA and by analyzing the editing of site-directed mutant RNAs in transfected cells and in cell extracts. Because editing occurs in the absence of viral antigens and the specificity for the HDV editing target site is present even in extracts from Drosophila cells, it is likely that HDV RNA is edited by one or more cellular factors that are conserved among higher eukaryotes. These results raise the likelihood that double-stranded RNA adenosine deaminase specifically edits HDV antigenomic RNA.

Adenosine↗
PubMed1994

Radioimmunotherapy of metastatic colorectal tumours with iodine-131-labelled antibody to carcinoembryonic antigen: phase I/II study with comparative biodistribution of intact and F(ab')2 antibodies.

Studies in animal tumour models of colorectal cancer suggest that F(ab')2 antibody fragments to carcinoembryonic antigen (CEA) labelled with iodine-131 give superior therapy compared with intact anti-CEA antibody. The purpose of this study was to investigate this hypothesis in patients. Ten patients received intact A5B7 IgG1 mouse monoclonal antibody (MAb) to CEA and nine patients received the F(ab')2 fragment of the same antibody. The biodistribution for each molecule was compared using quantitative single-photon emission computerised tomographic (SPECT) gamma-camera imaging. Tumour responses were seen in both groups and myelosuppression was the limiting toxicity. F(ab')2 localised more rapidly than intact antibody in tumour, giving a mean percentage injected activity per kg at 4.25 h after injection of 8.2% for F(ab')2 compared with 4.4% for intact antibody (P < 0.05). No significant difference in antibody clearance from, or cumulative dose per unit administered activity (cGy MBq-1) to, tumour was seen. Distribution in blood was similar for both the intact and fragment antibody. These findings are consistent with more rapid penetration of the smaller F(ab')2 into tumour masses. More efficient early uptake will give higher maximum dose rates to the tumour which is valuable for radioimmunotherapy (RIT) when low dose rates may limit effectiveness of treatment. F(ab')2 fragments may provide a substantially enhanced method of delivering RIT.

Adult↗
PubMed1993

A genotype of hepatitis D virus that occurs in northern South America.

Hepatitis D virus (HDV) is the cause of an unusually severe form of liver disease with distinct histologic features (morula cell) that occurs throughout northern South America and certain other areas of the world. Clinical studies of HDV disease worldwide indicate that there is, in fact, a wide variation in pathogenesis, and the reasons for these differences are presently unknown. One possible explanation is that factors associated with the viral genotype are determinants of HDV pathogenesis. In this study, nucleic acid sequences were determined for three different northern South American HDV isolates which were obtained from individuals with severe disease or a family history of severe disease, in areas that are hyperendemic for this disease pattern. The sequences of these three isolates are very similar to one another but only distantly related to other published HDV sequences. Comparison of the sequence of a semiconserved region from a total of 14 isolates indicates that there are at least three HDV genotypes. Most published HDV sequences, including those from North America, Europe, the Middle East, the South Pacific, and Asia, belong to a single genotype which may have some geographically based subtypes. A single Japanese isolate is the sole representative of a second HDV genotype. The South American sequences reported here constitute a third genotype. The association of a particular genotype with the severe form of type D hepatitis that occurs in northern South America supports the hypothesis that HDV genetic factors are important determinants in the pathogenesis of type D hepatitis.

Adolescent↗
PubMed1992

Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism.

Hepatitis delta virus (HDV) nucleotide 1012 is edited from uridine to cytidine in 10-40% of the RNA genomes during replication. This editing event is an important control point in the HDV life cycle because it results in both the packaging of viral RNA and the inhibition of HDV replication. We find that the editing event is highly specific for both the sequences neighboring nucleotide 1012 and the base-paired context of position 1012 within the unbranched rod structure of HDV RNA. Prior studies identified the base transition at nucleotide 1012 but were unable to distinguish between editing of the genomic versus the antigenomic strands [Luo, G. X., Chao, M., Hsieh, S. Y., Sureau, C., Nishikura, K. & Taylor, J. (1990) J. Virol. 64, 1021-1027]. In this study, comparisons of mutations that differentiate between base pairing in genomic and antigenomic RNAs indicate that the genomic strand of HDV is the actual editing substrate. We conclude that the virus uses a uridine to cytidine editing mechanism, which is provided by the host cell.

Animals↗
PubMed1991

Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase.

Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluoro-dUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human beta-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.

Animals↗
PubMed1991

Translation and the stability of mRNAs encoding the transferrin receptor and c-fos.

Turnover of the full-length human transferrin receptor (TfR) mRNA is regulated by iron, and this regulation is mediated by the transcript's 3' untranslated region. Alterations in the sequence of the TfR mRNA regulatory region have been identified that render the mRNA unregulated by iron and intrinsically unstable. When cells expressing this unstable mRNA are treated with inhibitors of protein synthesis (cycloheximide or puromycin), the steady-state level of the encoded human TfR mRNA is increased due to a stabilization of the transcript. A similar set of observations has been made using a chimeric mRNA in which the rapid turnover determinant of the TfR mRNA is replaced by the (A+U)-rich region from the 3' untranslated region of c-fos mRNA. To distinguish between a labile protein participant in the degradation of these mRNAs and a requirement for their translation per se, we introduced a ferritin iron-responsive element into the 5' untranslated region of each of these mRNAs. The presence of the 5' iron-responsive element allowed us to use iron availability to alter the translation of the mRNAs in question without global effects on cellular protein synthesis. Although specific translation of these mRNAs could be inhibited by iron chelation to a degree comparable to that seen with cycloheximide (approximately 95% inhibition), no effects on mRNA turnover were observed. These data support a model in which a trans-acting labile protein is necessary for the turnover of these mRNAs rather than there being a requirement for the translation of the mRNAs themselves.

Animals↗