Sequence map of the 2 Mb Giardia lamblia assemblage A chromosome.

The gut protozoan parasite, Giardia lamblia (Assemblage A), has 5 major chromosomes, 1 of which is 2 Mb, as determined from gel separations of whole chromosomes. We originally published a physical map of this chromosome and, now, using the sequence data from 46 chromosome-specific probes, have produced a sequence map of the 2 Mb chromosome. Comparison of the probe sequences with the Giardia genome database (http://GiardiaDB.org) has identified 4 scaffolds (CH991771, CH991780, CH991782, and CH991767) belonging to the 2 Mb, Assemblage A, chromosome. Because of the density of probe sequences, we have been able to predict the orientation of the scaffolds and have identified erroneous inclusions in scaffold CH991767. Exclusion of erroneously included sequences resulted in a 1.96 Mb chromosome sequence. This study brings together experimental data and the GiardiaDB data to compile the sequence of a whole chromosome.

Epstein-Barr virus-encoded RK-BARF0 protein expression.

The cellular localization of the Epstein-Barr virus-encoded RK-BARF0 protein was analyzed by fluorescence microscopy and immunoblotting. The recombinant RK-BARF0 protein was found to be tightly bound to nuclear structures, whereas 16- to 20-kDa RK-BARF0 derivatives, generated by differential splicing of the RK-BARF0 transcript, were present throughout the cell. Moreover, a previously generated anti-RK-BARF0 rabbit serum was found to cross-react with cellular proteins, showing that the previously identified 30- to 35-kDa membrane-associated proteins do not represent RK-BARF0.

A-type and B-type Epstein-Barr virus differ in their ability to spontaneously enter the lytic cycle.

In this study replication of A-type and B-type Epstein-Barr virus (EBV) strains has been assessed. A-type and B-type type lymphoblastoid cell lines (LCLs) were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence of viral capsid antigens (VCA) in these LCLs was determined by immunofluoresence assay and by immunoblotting. All of the B-type EBV strains were capable of spontaneously generating virus regardless of the origin of the donor cells. In contrast the A-type strains, other than strain IARC-BL36, did not readily produce VCA in any of the different donor lymphocytes used. This study demonstrates another biological difference between the two virus types: their ability to spontaneously enter the lytic cycle.

A functional link for major TCR expansions in healthy adults caused by persistent Epstein-Barr virus infection.

Dramatic clonal expansions of unknown functional significance have been documented in the T cell receptor (TCR) alpha beta peripheral blood repertoires of apparently healthy adults. In this study, we provide evidence that persistent infection with the ubiquitous Epstein-Barr virus (EBV) causes major distortions within the memory repertoire of healthy virus carriers. Using complementarity determining region 3 (CDR3) length analysis to measure repertoire diversity, dominant expansions that dramatically skewed the entire TCRBV6 blood repertoire towards oligoclonality were enriched in the CD8(+)CD45RO+CD45RA- subset of HLA B8(+) healthy virus carriers. Evidence of phenotypic heterogeneity between individuals was also observed for these expansions based on their variable coexpression of CD45RO and CD45RA. TCR junctional region sequencing revealed that these expansions were clonal and that they represented commonly selected HLA B8-restricted memory cytotoxic T cells that recognize the immunodominant latent EBV epitope, FLRGRAYGL. Furthermore, the functional identity of these virus-specific CD8(+) T cells was confirmed by their FLRGRAYGL-specific cytotoxicity. Therefore, the functional significance of dramatic clonal expansions in healthy adults can be linked in some cases to virus-specific CD8(+) T cells that play an essential role in immunosurveillance. This first identified link for expansions in the circulation of healthy adults strongly implies that restricted-memory TCR responses to environmental antigens play a pivotal role in expansion development, which should have an important impact on studies interpreting TCR expansion patterns in health and disease.

Regulation of interleukin-1beta transcription by Epstein-Barr virus involves a number of latent proteins via their interaction with RBP.

Epstein-Barr virus (EBV) infects B cells, resulting in the outgrowth of immortalised lymphoblastoid cell lines (LCLs). Here, we demonstrate through the use of intracellular staining that interleukin-1beta (IL-1beta) is expressed in LCLs and investigate the influence of the individual latent proteins on the expression of IL-1beta. Using RT-PCR, IL-1beta was shown to be up-regulated in EBV-transformed LCLs as well as in group III Burkitt's lymphoma (BL) cell lines, compared with group I BL cell lines. The up-regulation of IL-1beta message could be mediated by the latent membrane protein-1, EBV nuclear proteins 2, 3, 4, and 6 genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that the -300 region of the IL-1beta promoter, which contains a nuclear factor-kappaB (NF-kappaB) binding site, contained a functional RBP binding site. Binding of RBP to this site could be inhibited by addition of EBV nuclear proteins 3 and 6, suggesting that these proteins displace RBP from its recognition sequence, removing transcriptional repression and allowing gene transcription to occur. In group I BL cells, containing low levels of NF-kappaB, only RBP binding was observed in EMSAs, whereas NF-kappaB binding could be demonstrated in EBV-transformed B cell lines containing high levels of activated NF-kappaB. In addition, the expression of latent membrane protein-1 led to activation of NF-kappaB that was capable of binding the IL-1beta promoter. The study demonstrates that EBV can up-regulate IL-1beta expression, possibly by using RBP, NF-kappaB, or both.

Interferon-resistant human melanoma cells are deficient in ISGF3 components, STAT1, STAT2, and p48-ISGF3gamma.

The mechanism of IFN resistance was examined in three long-term cell lines, SK-MEL-28, SK-MEL-3, and MM96, exhibiting significant variation in responsiveness to the antiproliferative and antiviral effects of type I IFNs. The JAK-STAT components involved in IFN signal transduction were analyzed in detail. After exposure to IFN, activation of the IFN type I receptor-linked tyrosine kinases, JAK-1 and TYK-2, was detected at similar levels in both IFN-sensitive and IFN-resistant cell types, indicating that IFN resistance did not result from a deficiency in signaling at the level of receptor-associated kinase activation. However, analysis of ISGF3 transcription factor components, STAT1, STAT2, and p48-ISGF3gamma, revealed that their expression and activation correlated with cellular IFN responsiveness. The analysis was extended to also include IFN-sensitive primary melanocytes, three additional IFN-resistant melanoma cell lines, and seven cell cultures recently established from melanoma patient biopsies. It was consistently observed that the most marked difference in ISGF3 was a lack of STAT1 in the resistant versus the sensitive cells. Transfection of the IFN-resistant MM96 cell line to express increased levels of STAT1 protein partially restored IFN responsiveness in an antiviral assay. We conclude that a defect in the level of STAT1 and possibly all three ISGF3 components in IFN-resistant human melanoma cells may be a general phenomenon responsible for reduced cellular responsiveness of melanomas to IFNs.

Comparison of the biological properties of two anti-mucin-1 antibodies prepared for imaging and therapy.

A comparison was made between the murine anti-MUC1 antibody BC2 (which reacts with the peptide epitope APDTR) and the "humanised" antibody hCTMO1 from CellTech, which reacts with the MUC1 epitope RPAP. Preliminary studies demonstrated that hCTMO1 was a "good" antibody whereas BC2 was not. Various parameters were determined and conclusions reached. (a) Affinity: the affinity of hCTMO1 was 2.60 x 10(7) M(-1) and that of BC2 was 1.36 x 10(7) M(-1); we did not consider these numbers to be substantially different, although hCTMO1 was clearly of higher affinity than BC2. (b) On/off rate at 4 degrees C: both antibodies bound effectively to the MUC-1 transfectant MOR5-CF2; the association rate for hCTMO1 was 3.8 times that of BC2 and the dissociation rate for BC2 was twice as fast as that of hCTMO1. (c) On/off rates at 37 degrees C: at 37 degrees C the association rate for hCTMO1 was greater than that of BC2. (d) Internalization: hCTMO1 was also more efficient at internalising bound antibody; 70% of bound hCTMO1 was internalised, whilst 6% of bound BC2 was internalised. From these studies it was clear that, while hCTMO1 was of similar affinity to BC2, the faster uptake and internalisation and lower off rate indicated that it was likely to be a superior antibody; this was proven in vivo. (e) Localisation: hCTMO1 bound much better in vivo than BC2 (68% compared to 28%). (f) Therapeutic experiments: BC2-idarubicin conjugates were essentially ineffective in eradicating tumours in mice whereas hCTMO1-idarubicin had a dramatic effect on breast cancer tumour cells growing in mice. We conclude that the simple measurements on/off rates and internalisation at 37 degrees C are the most important parameters to use to determine antibody effectiveness, prior to embarking on clinical studies.

Both A type and B type Epstein-Barr virus nuclear antigen 6 interact with RBP-2N.

Using the yeast two-hybrid system, Epstein-Barr virus nuclear antigen 6A (EBNA6A) was found to interact with the RBP-2N isoform of RBP-J kappa. The interaction of EBNA6A and EBNA6B with RBP-2N was compared and the results indicated that EBNA6B was less efficient at interacting with RBP-2N than was EBNA6A. Deletion mutation analysis of EBNA6A identified a region involved in the interaction with RBP-2N, while analysis of RBP-2N identified a domain which interacts with EBNA6A. The region of RBP-2N to which EBNA6A binds has previously been shown to interact with EBNA2.

Epstein-Barr nuclear antigen-3 and -4 interact with RBP-2N, a major isoform of RBP-J kappa in B lymphocytes.

The Epstein-Barr nuclear antigen (EBNA)-3 and EBNA-4 proteins are thought to act as transcriptional transactivators. The yeast two-hybrid system and coimmunoprecipitation were used to demonstrate that EBNA-3 and -4 associate with the DNA-binding protein RBP-2N, an isoform of RBP-J kappa. A comparison between EBNA-3, EBNA-4, and EBNA-6 binding to RBP-2N indicated that EBNA-3 enhanced beta-galactosidase activity 4-fold more than EBNA-6 and 30-fold more than EBNA-4. Assay of RBP-2N deletion mutants demonstrated that EBNA-3 binds to regions of RBP-2N which are distinct from those to which EBNA-2 and -6 interact, whereas EBNA-4 binds to the same region of RBP-2N as EBNA-2 and -6 (amino acids 159-331 of RBP-2N). Interaction of both A- and B-type EBNA-3 with RBP-2N was also demonstrated by immunoprecipitation. RT-PCR analysis of a panel of B cell lymphomas and lymphoblastoid cell lines demonstrated that higher levels of RBP-2N were expressed, in comparison to RBP-J kappa, indicating that RBP-2N is a major isoform expressed in B cells. These results suggest that all the EBNA-3 family proteins lead to transcriptional regulation via interaction with RBP-2N.

Mitochondrial respiratory chain inhibitors induce apoptosis.

In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycin A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells within 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

Antibody-targeted drugs for the therapy of cancer.

The advent of monoclonal antibodies has revitalised the concept of magic bullets and various agents (eg. drugs, toxins and isotopes) have been conjugated to monoclonal antibodies for selective delivery to tumours. Preclinical studies in mouse tumour models have been impressive and have lead to several clinical trials. These phase I trials have been less impressive. However, keeping in mind the aim of Phase I trials, the safety of using these conjugates in humans have been established. Several, major problems still remain to be overcome before these agents may be useful for the treatment of cancer. These problems stem from the nature of tumour vasculature, cytotoxic activity of the moiety linked to antibody and the targeted tumour antigen expressed on the cell surface. This review will deal with these various aspects described above and possible approaches to overcome these obstacles with a definite bias towards drug-monoclonal antibody conjugates. However, these concepts are equally applicable for improved targeting of other agents.

Immunochemotherapy of human colon carcinoma xenografts in nude mice using combinations of idarubicin-monoclonal antibody conjugates.

Tumour cell heterogeneity is probably a principal cause of treatment failure and represents a formidable barrier for effective antibody-targeted chemotherapy. Idarubicin (Ida), a more potent and less cardiotoxic analogue of daunomycin, has been demonstrated to specifically target and eradicate homogeneous, cloned, murine tumour cell populations in vitro and in vivo when coupled to monoclonal antibodies (MoAb); however, the antitumour activity of Ida-MoAb conjugates against human tumour xenografts remains to be established. In this study, the value of cotargeting conjugates to different human tumour-associated antigens within a solid tumour has been assessed by comparing the effects of combinations of Ida-anti-colon carcinoma MoAb conjugates with any one Ida-anti-colon carcinoma MoAb conjugate used alone. Individual Ida-MoAb conjugates have previously been evaluated for their specific binding and cytotoxicity to one of two different human colon carcinoma xenografts (Colo 205 or LIM2210) in vitro, although their efficacy alone or in combination required assessment in vivo. Combinations of the most effective Ida-MoAb conjugates were demonstrated to enable a greater number of complete tumour regressions than the most efficacious Ida-MoAb conjugate administered alone in vivo; some combinations inhibited control tumour growth by up to 95%. This study suggests that Ida-MoAb conjugates can be effective against subcutaneous human tumours in nude mice, although it is unlikely that any single conjugate will eradicate all the tumour cells in a solid tumour, and the value of 'cocktails' of drug-MoAb conjugates against some xenografts (i.e. LIM2210) appears to be limited.

The comparative effects of IL-1 and TNF on AMN-anti-Ly 2.1 immunoconjugate therapy.

The administration of mTNF alpha and hIL-1 alpha was investigated for their potential to increase the anti-tumor activity of AMN-anti-Ly-2.1 against the Ly-2.1+ murine thymoma ITT(1) 75 NS E3. Dose response studies using mTNF alpha alone demonstrated a single 10 micrograms iv injection produced 30% inhibition in tumor size while 3 doses of 1 microgram administered on alternative days produced 70% tumor inhibition. By contrast, hIL-1 alpha was unable to significantly reduce E3 tumor size using single doses up to 10 micrograms or a total of 30 micrograms administered in 3 doses (iv or ip). However, intratumor injection of hIL-1 alpha (20 micrograms injected in 2 doses) produced 20% inhibition in tumor size. Combination therapy using AMN immunoconjugates with mTNF alpha showed enhanced antitumor activity compared to each agent alone. Biodistribution studies revealed that anti-tumor activity, was due to increased localization (2-3 fold) of AMN immunoconjugates in the presence of mTNF-alpha whereas huIL-1 alpha was without effect unless accompanying toxicity was seen. Clearly for this tumor, mTNF alpha potentiated the effects of AMN immunoconjugates. Despite the shared biological properties of these cytokines, mTNF alpha is superior to hIL-alpha for augmenting drug immunoconjugate.

Antitumor effect of 2'-deoxy-5-fluorouridine conjugates against a murine thymoma and colon carcinoma xenografts.

The conjugation of antineoplastic drugs to monoclonal antibodies reactive with tumor associated antigens conveys selective cytotoxicity, overcoming the systemic toxicities caused by drugs during standard chemotherapy. 2'-Deoxy-5-fluorouridine, a more potent derivative of 5-fluorouracil, is an antimetabolite which exerts its cytotoxic action by inhibiting the enzyme thymidylate synthetase and as a result inhibits DNA synthesis. 2'-Deoxy-5-fluorouridine was successfully conjugated to anti-Ly-2.1 reactive with the murine thymoma ITT(1)75NS E3, I-1, and 250-30.6 reactive with human colon cancer cells using the active ester of 2'-deoxy-5-fluoro-3'-O-succinoyluridine (5FdUrdsucc). In vitro, 5Fd-Urdsucc-anti-Ly-2.1 was selectively cytotoxic against ITT(1)75NS E3 murine thymoma cells at nanomolar concentrations. The human colon carcinoma cell LIM1899 was inhibited by 5FdUrdsucc-I-1 conjugates in the range of 10(-7)-10(-8) M, as were Colo 205 cells by 5FdUrdsucc-250-30.6 conjugates. In vivo, 5FdUrdsucc conjugates were more effective than equivalent amounts of free 5FdUrdsucc. Against the ITT(1)75NS E3 murine thymoma, a single dose of 100 micrograms (5FdUrdsucc equivalents) 5FdUrdsucc-anti-Ly-2.1 resulted in 85% tumor inhibition compared to mean tumor size of control mice. Irrelevant 5FdUrdsucc conjugates failed to inhibit tumor growth. Multiple doses of 5FdUrdsucc-I-1 conjugate produced 50% growth inhibition of the moderately differentiated tumor LIM1899. In contrast, the human colon carcinoma Colo 205 was relatively resistant to free 5FdUrdsucc and 5FdUrdsucc-250-30.6 conjugates.

In vitro antitumor activity of 2'-deoxy-5-fluorouridine-monoclonal antibody conjugates.

5-Fluorouracil (5-FU) is an anticancer drug used in patients for the treatment of gastric and breast cancer and used either alone or in combination with methotrexate is one of the few drugs with some effect on colon cancer. 2'-Deoxy-5-fluorouridine (5-FUdr) (1) is an analogue based on 5-FU and can be covalently linked to a murine anti-Ly-2.1 monoclonal antibody (mAb) with the active ester derivative of 2'-deoxy-5-fluoro-3'-O-(carboxypropanoyl)uridine (5-FUdr-succ) (4). Such immunoconjugates can contain up to 42 residues of drug, although the most antibody activity was retained when substitution ratios were between 10 and 25 molecules of drug to mAb. In a cytotoxicity assay, 50% inhibition of [3H]deoxyuridine incorporation (IC50) with a murine Ly-2.1+ve thymoma cell line was 6 nM for 5-FUdr-anti-Ly-2.1, which is 12-fold more than that for free 5-FUdr (IC50 = 0.51 nM) but similar to that of 5-FUdr-succ (IC50 = 5.2 nM). The 5-FUdr-monoclonal antibody conjugates (5-FUdr-mAb) were 100-fold more active on the Ly-2.1+ve E3 cell line than on the Ly-2.1-ve BW5147 OU- cell line. The high in vitro activity and specificity of 5-FUdr-MoAb conjugates indicates that potent in vivo activity of these conjugates should be expected.

Effect of tumor necrosis factor on the antitumor efficacy and toxicity of aminopterin-monoclonal antibody conjugates: parameters for optimization of therapy.

Immunoconjugates of monoclonal antibodies with drugs, isotopes, or toxins are currently being investigated for their therapeutic effect on tumors. However, all have problems of access of the immunoconjugate to the tumor, particularly with solid tumors. To address this problem, we have used aminopterin-monoclonal antibody (AMN-mAb) conjugates combines with murine tumor necrosis factor (mTNF-alpha), which is known to have specific effects on tumor vasculature. In a murine model, well-established tumors (measuring 1.0-1.4 cm in diameter) were either totally eradicated or considerably reduced in size with combined therapy--a greater effect than with either mTNF-alpha or AMN-mAb used alone. The mechanisms involved in the improved antitumor effect were investigated using in vitro assays, autoradiography, and biodistribution experiments. mTNF-alpha was found both to increase the cytotoxic activity of the conjugate in vitro and to increase in vivo tumor localization of mAb up to 5-fold. The timing of mTNF-alpha administration was crucial to effects on tumor localization; mTNF-alpha given with mAb caused the greatest increase in localization and mTNF-alpha given well before mAb decreased localization. mTNF-alpha also reduced the toxicity to mice of AMN-mAb depending on the timing of injection. These results indicate that mTNF-alpha has a useful role in potentiation of immunoconjugate therapy but shows the need for careful planning of the dose regimen.