Circulating tumour cells--a bona fide cause of metastatic cancer.

Circulating tumour cells (CTCs) are emerging as important prognostic markers and have potential clinical utility as tumour biomarkers for targeted cancer therapy. Although CTCs were proposed more than 100 years ago as potential precursors that may form metastatic lesions, formal evidence that CTCs are indeed capable of initiating metastases is limited. Moreover, the process of CTCs shedding into the circulation, relocating to distant organ sites and initiating metastatic foci is complex and intrinsically inefficient. To partially explain the metastatic process, the concepts of CTCs as metastatic precursors or pre-metastatic conditioners have been proposed; however, it is questionable as to whether these are both variable pathways to metastasis or just markers of metastatic burden. This review explores the evidence for CTCs in the initiation and progression of metastatic cancer and the data supporting these different concepts in an attempt to better understand the role of CTCs in metastasis. A greater understanding of the metastatic potential of CTCs will open new avenues for therapeutic interventions in the future.

Circulating tumour cells and circulating free nucleic acid as prognostic and predictive biomarkers in colorectal cancer.

The detection of circulating tumour cells or circulating free tumour nucleic acids can potentially guide treatment and inform prognosis in colorectal cancer using minimally invasive "liquid biopsies". Current literature supports the notion that high circulating tumour cell counts or presence of tumour nucleic acid correlate with inferior clinical outcomes for patients, but they are not yet part of routine clinical care. Future research evolves around the examination of the molecular phenotype of circulating tumour cells. The key unanswered areas include differentiating between circulating tumour cell presence and their proliferative capacity and dormancy, identifying tumour heterogeneity and understanding the epithelial-mesenchymal transition.

The duplicitous effects of interleukin 4 on tumour immunity: how can the same cytokine improve or impair control of tumour growth?

Successful tumour immunity relies on innate and adaptive immune responses, with cytokines like interleukin 4 (IL-4) known to influence tumour clearance in both positive and negative ways. Here, we summarise some of the murine tumour models used over the past two decades to assess the impact of IL-4 on tumour immunity, with emphasis on the effects of IL-4 on the tumour-induced CD8 T-cell response. These data are compared with our own recent studies showing that IL-4 impairs CD8+ T-cell-mediated immunity against the mastocytoma cell line P815 expressing the immunogenic HLA-CW3 gene; moreover, we hypothesise that quantitative and qualitative differences in the HLA-CW3-induced CD8+ T-cell response impair control of tumour growth and aid the development of secondary tumours. We conclude that the duplicitous effects of IL-4 on tumour immunity depend on the type of effector cell (adaptive/innate) mediating tumour clearance and whether tumour growth depends on stromal infrastructure. Thus, the search for factors that improve or weaken the effectiveness of tumour-specific T cells has to be continued to improve modern approaches of immunotherapy against cancer.

Murray Valley encephalitis: case report and review of neuroradiological features.

We report on a child with diffuse symmetrical thalamic enlargement and signal increase on MRI, representing changes caused by Murray Valley encephalitis (MVE). Very little has previously been reported on the neuroradiological findings of MVE, also known as Australian encephalitis. It is endemic to tropical North Australia, particularly Western Australia and the Northern Territory, but can occur in other parts of Australia. The last epidemic was in south-eastern Australia in 1974. Australian encephalitis is the second most serious acute viral encephalitis to be encountered in Australia. Clinicians need to be aware of MVE in this era of ever-increasing travel. Our aim is to highlight these finding and further define the neuroradiological features.

Antiserum raised against the Epstein-Barr virus BARF0 protein reacts to HLA-DR beta chain.

The role that Epstein-Barr virus plays in nasopharyngeal carcinoma and Burkitt's lymphoma has been under intense study for many years. With only a limited set of viral genes being expressed in these tumours it has been difficult to understand how the virus could cause/aid in the generation of the tumours. In 1997 a paper was published by Fries et al. [Fries et al. (1997) Identification of a novel protein encoded by the BamH1 A region of the Epstein-Barr virus. J Virol 71: 2765-2771.] in which a rabbit serum was generated and used to identify protein products (RK-BARF0) encoded from the BamH1 A region of EBV. In this paper we have isolated these proteins from two-dimensional gels and identified them, using mass spectrometry, as components of HLA DR.

Chryseobacterium in burn wounds.

Chryseobacteria are gram negative organisms, formerly known as Flavobacteria, which rarely cause infections of burn wounds. This article documents three cases of Chryseobacterium infection in burn wounds and adds to the other two cases that have been reported in English literature. Two patients died, with one of the deaths linked to a Chryseobacteria bacteraemia. In two patients, there was an associated history of first aid treatment with untreated water. Patients whose burn wounds are suspected to be infected with Chryseobacterium require wound excision and coverage in combination with antibiotic therapy such as ciprofloxacin, vancomycin and rifampicin.

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters.

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)-mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells. In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone. These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity.

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.

Differential splicing of antigen-encoding RNA reduces endogenous epitope presentation that regulates the expansion and cytotoxicity of T cells.

The activation of CTLs is dependent on the recognition of MHC-bound peptide present on the surface of APCs. We give evidence in this study that differential splicing of Ag-encoding RNA can decrease the antigenic dose in APCs and regulate the recall of human memory CTLs. Differential splicing of RNA that encoded an immunodominant HLA-B8-restricted CTL epitope of EBV reduced the functional presentation of this epitope, and consequently the in vitro expansion and activity of CTLs, as measured by MHC/peptide-tetramer staining and cytotoxicity assays. The reduced activity of the stimulated CTLs was not only due to lower numbers of Ag-specific CTLs but, surprisingly, was also characterized by decreased cytotoxicity of the CTLs to target cells presenting limiting amounts of the peptide epitope. As indicated by TCR repertoire analysis, the reduction in CTL activity was not caused by stimulation of distinct populations of TCR clonotypes. This study demonstrates how a common eukaryotic posttranscriptional mechanism of gene regulation can modulate the endogenous presentation of Ag and ultimately contribute to the fine tuning of immunological memory cells, which are important in the fight against pathogens and tumors and in autoimmunity.

Aeromonas wound infection in burns.

Infection of burn patients with the Aeromonas organism is an uncommon event. This paper documents four cases of Aeromonas hydrophila and one case involving both A. hydrophila and A. caviae occurring in burn patients between 1990 and 1998 at the Royal Brisbane Hospital burns unit. The organism was isolated from either skin swabs, tissue samples, blood cultures or cultured lines. In all patients there was a history of immersion in water immediately post burn. There is one case of invasion and destruction of deeper tissues and one fatality. Appropriate management requires a high index of suspicion if a history of immersion in untreated water post burn is given and the treatment involves aggressive excision and antibiotic therapy.

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.

Cutting edge: silencing virus-specific cytotoxic T cell-mediated immune recognition by differential splicing: a novel implication of RNA processing for antigen presentation.

Persistent viruses have developed potent strategies to overcome host immune defenses. In particular, viral interference with Ag presentation by HLA class I molecules can effectively impair the host's CTL function. Here we provide evidence for a novel aspect of differential splicing on endogenous processing of a latent viral transcript resulting in dominant protein isoforms from which the CTL determinant has been deleted. Consequently, virus-infected cells expressing these isoforms were poorly recognized by CTLs. Molecular analysis revealed that this splicing significantly reduced expression of the viral transcript encoding the relevant epitope to levels below the threshold required for CTL recognition. The importance of splicing was further reinforced by the observation of efficient CTL recognition of target cells expressing a truncated viral transcript that abolished differential splicing. Thus, differential splicing, which is a common mechanism of gene regulation in many pathogens, may unexpectedly interfere with immune recognition.

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.

Intron retention may regulate expression of Epstein-Barr virus nuclear antigen 3 family genes.

The nuclear antigen 3 family genes (EBNA-3, EBNA-4, and EBNA-6) of Epstein-Barr virus (EBV) are important for EBV-induced immortalization and survival of B lymphocytes. However, little is known about how the expression of these genes is regulated. Each of the EBNA-3, EBNA-4, and EBNA-6 genes consists of two exons separated by a small intron. Reverse transcriptase PCR assays revealed that the vast majority of the EBNA-3, EBNA-4, and EBNA-6 mRNA, expressed in transfected and EBV-infected B cells, retained intron sequences. Northern blot and S1 protection assays confirmed that most of the EBNA-3 mRNA contained intron. Examination of deletion mutants of EBNA-3 indicated that the EBNA-3 protein was not necessary for intron retention and that there was no splicing silencing element encoded in the EBNA-3 mRNA. Cell fractionation and RNA gradient analysis revealed that the unspliced EBNA 3 family mRNAs were transported into the cytoplasm and associated with the polysomes. However, Western blot analysis of FLAG-epitope tagged EBNA-3 gave no indication of the presence of splice variant protein forms of EBNA-3. In contrast, transiently transfected cells expressing EBNA-3 revealed a sixfold increase in EBNA-3 protein expression from the genomic EBNA-3 gene compared to EBNA-3 cDNA. These data show that the intronic sequences can influence EBNA-3 protein expression and suggest that intron retention may provide a means for the fine-tuning of expression of the individual EBNA 3 family genes.

Identification of a cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression.

The Epstein-Barr virus (EBV)-encoded BARF0 open reading frame gene products are consistently expressed in EBV-positive Burkitt's lymphoma (BL) cell lines, nasopharyngeal carcinoma cell lines, and lymphoblastoid cell lines (LCLs). Here we show that the BARF0 sequence includes an HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope. By using theoretically predicted HLA A2 binding motifs and peptide-loaded antigen presentation-deficient T2 cells, polyclonal BARF0-specific CD8(+) CTLs were isolated from four different healthy EBV-seropositive donors but not from two seronegative donors. These CTL lines recognized the peptide epitope LLWAARPRL, which was found to be conserved in 33 of 34 virus strains originating from Caucasian, African, and Asian individuals. The BARF0-specific CTL lines could lyse EBV-negative BL cells stably transfected with the BARF0 gene but did not kill HLA A2-matched EBV-positive BL cells and LCLs in a standard 51Cr release assay. Reverse transcriptase PCR analysis demonstrated that these EBV-positive cell lines expressed significantly lower levels of BARF0 mRNA than transfected cells. This data indicated that the BARF0 epitope could be endogenously processed; however, antigen levels in the target cell were a limiting factor for the effective interaction between BARF0-expressing cells and CTLs. The limited expression of BARF0 antigen in EBV-infected BL cells and LCLs might contribute to the escape of immune recognition from virus-specific CTLs present in the host.

Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization.

Human immunodeficiency virus (HIV) Nef functions are thought to be mediated via interactions with cellular proteins. Utilizing zone velocity sedimentation in glycerol gradients we found that recombinant HIV-1 Nef non-covalently associates with actin forming a high-molecular-mass complex of 150-300 kDa. This Nef/actin complex was present in human B and T lymphocytes but not in insect cells and was dependent on the N-terminal myristoylation of Nef, whereas the SH3-binding proline motif of Nef was not involved. Despite being myristoylated, HIV-2 Nef did not associate with actin. This might reflect differences in the subcellular localization of Nef since cell-fractionation experiments revealed that HIV-1 Nef was virtually exclusively localized in the cytoskeletal (detergent-insoluble) fraction whereas HIV-2 Nef had significantly reduced affinity for the cytoskeleton. Colocalization experiments in HIV-1-infected CD4+ fibroblasts revealed that Nef/actin complexes may also exist in HIV-infected cells. This novel interaction of HIV-1 Nef with actin provides insight into the association of Nef with cellular structures and reveals general differences in the interactions of the Nef proteins from HIV-1 and HIV-2.

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.

Engagement of CD40 antigen with soluble CD40 ligand up-regulates peptide transporter expression and restores endogenous processing function in Burkitt's lymphoma cells.

Cells from the EBV-associated tumor, Burkitt's lymphoma (BL), are known to be highly inefficient at endogenous processing of class I-restricted CTL epitopes due to a consistent loss of peptide transporters (TAP) and MHC expression. We investigated the potential of CD40 engagement to up-regulate the expression of class I-processing genes and to enhance the immunogenicity of these malignant cells toward EBV-specific CTLs. Here we show that engagement of CD40 Ag with soluble CD40 ligand (CD40L) up-regulates TAP-1 and HLA class I expression on BL cells. More importantly, analysis of the Ag-processing function, using a recombinant vaccinia virus to transiently express the EBV nuclear Ags, revealed that CD40L-treated BL cells consistently processed endogenously synthesized viral Ags for recognition by HLA class I-restricted, virus-specific CTLs. These findings raise the possibility that CD40L treatment of tumor cells might be exploited in immunotherapeutic protocols.