David R. Fitzpatrick

A Critical Role for Dnmt1 and DNA Methylation in T Cell Development, Function, and Survival

The role of DNA methylation and of the maintenance DNA methyltransferase Dnmt1 in the epigenetic regulation of developmental stage- and cell lineage-specific gene expression in vivo is uncertain. This is addressed here through the generation of mice in which Dnmt1 was inactivated by Cre/loxP-mediated deletion at sequential stages of T cell development. Deletion of Dnmt1 in early double-negative thymocytes led to impaired survival of TCRalphabeta(+) cells and the generation of atypical CD8(+)TCRgammadelta(+) cells. Deletion of Dnmt1 in double-positive thymocytes impaired activation-induced proliferation but differentially enhanced cytokine mRNA expression by naive peripheral T cells. We conclude that Dnmt1 and DNA methylation are required for the proper expression of certain genes that define fate and determine function in T cells.

Transforming Growth Factor-Beta: Antisense RNA-Mediated Inhibition Affects Anchorage-Independent Growth, Tumorigenicity and Tumor-Infiltrating T-Cells in Malignant Mesothelioma

Transforming growth factor-beta (TGF-beta) is produced by a number of tumor cell types including human malignant mesothelioma (MM), but its role as a direct or indirect factor in tumorigenesis is incompletely understood. We have investigated the expression of TGF-beta isoforms by human and murine MM cells and have analysed the effects of inducible antisense RNA-mediated inhibition of TGF-beta expression on murine MM in vitro and in vivo. The results showed that (a) TGF-beta 1 and -beta 2 were produced by both human and mouse MM cells, (b) antisense RNA against either TGF-beta 1 or -beta 2 cross-inhibited both TGF-beta 1 and -beta 2 expression, (c) inhibition of TGF-beta expression reduced the anchorage-independent growth of MM cells in vitro and the tumorigenicity of MM cells in vivo, and (d) inhibition of TGF-beta expression led to increased T lymphocyte infiltration into tumors. The data suggest that TGF-beta has multiple tumor-enhancing effects in MM.

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (Mphi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to Mphi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of, various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca(2+)/Mg(2+) enhanced DV binding. This argues against involvement of beta(2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains.

Patho- and immunobiology of malignant mesothelioma: characterisation of tumour infiltrating leucocytes and cytokine production in a murine model

Malignant mesothelioma (MM) is an aggressive, uniformly fatal serosal tumour, usually associated with asbestos exposure, for which there currently is no effective treatment. In order to gain insight into the mechanism(s) whereby MM might escape immune surveillance, a murine model for MM was used (a) to characterise the tumour-infiltrating lymphocytes (TIL) and macrophages (TIM) phenotypically, (b) to examine systemic immune recognition of MM, and (c) to examine the possible influence of tumour-derived cytokines on systemic and local pathobiological manifestations of MM. A profound down-regulation of lymphocyte surface markers, known to be infolved in T cell activation, was found in TIL. Likewise, although TIM were present in large numbers, their expression of MHC class II antigen and integrins was weak or absent, suggestive of altered functional activity. Significant amounts of cytokines, in particular transforming growth factor β, interleukin-6 (IL-6), IL-1 and tumour necrosis factor were produced during the course of MM tumour development-directly by the MM cells and/or indirectly in response to tumour growth. These factors may contribute both to derangement of antitumour effector mechanisms and to the clinical and pathological manifestations of the disease.

Interleukin-6 involvement in mesothelioma pathobiology: inhibition by interferon alpha immunotherapy

A role for interleukin-6 (IL-6) in malignant mesothelioma has been suggested by the clinically presenting symptoms of mesothelioma patients, which include fever, weight loss and thrombocytosis. A murine model of malignant mesothelioma was therefore used to examine the potential role of IL-6 in this cancer type and whether the effect of interferon α (IFNα) therapy on mesothelioma might be mediated, in part, by regulating IL-6 levels and/or IL-6-induced pathobiology. A panel of human and murine mesothelioma cell lines was assayed for endogenous IL-6 production in a bioassay, and for IL-6-mRNA expression. Four out of 5 human and 5 out of 15 murine mesothelioma cell lines produced moderate to high levels of bioactive IL-6 in vitro. This result was corroborated by mRNA detection. One of the representative murine cell lines, AB22, was chosen for further in vivo studies in the murine mesothelioma model. In AB22-inoculated mice detectable serum IL-6 levels were found to precede macroscopically detectable tumour growth, clinical signs (cachexia, abdominal distension, diarrhoea) and changes in the peripheral lymphoid organs (cell depletion and functional depression). Treatment with anti-IL-6 antibody curtailed the clinical symptoms (P<0.001), as did treatment with recombinant human (rhu) IFNα (P<0.001). Neither anti-IL-6 antibody nor rhuIFNα had a direct growth-inhibitory effect on the AB22 mesothelioma cell line in vitro, however, in vivo rhuIFNα treatment of mice inoculated with AB22 cells attenuated both IL-6 mRNA expression in the tumours and serum IL-6 levels, ameliorated the depression of lymphocyte activities, and enhanced the number of tumour-infiltrating lymphocytes and macrophages. On the basis of these results it is suggested that IL-6 mediates some of these effects, directly or indirectly, and that a combination therapy of rhuIFNα and anti-IL-6 antibody may be an improved palliative treatment for patients with malignant mesothelioma.

Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system

In a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289-424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum-free conditions, more than 95% of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue-3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.

Altered CD3 chain and cytokine gene expression in tumor infiltrating T lymphocytes during the development of mesothelioma

The mechanisms whereby tumors escape immunosurveillance remain poorly understood. De-activation or deviation of T lymphocyte responses may occur following exposure to tumor-associated or -derived signals. In the present study it is demonstrated that during development of syngeneic malignant mesothelioma in mice, the relative CD3 delta, CD3 gamma and CD3 zeta mRNA levels expressed by tumor infiltrating lymphocytes (TIL) decrease, while CD3 epsilon mRNA levels remain relatively constant. Expression of IFN gamma mRNA by TIL decreased during tumor development, while IL-2 mRNA levels showed slight increases. IL-3 mRNA was not detected at any time during tumor development and IL-4 transcripts were detected in the later stages of tumor development. In the spleens of tumor-bearing mice, IL-2 transcripts were detected throughout the time course from days 1 to 22(24), while IFN gamma mRNA was only detected at early times from days 0-13. Previous work demonstrated a role for tumor cell-derived TGF beta in the immunobiology of mesothelioma. Here it is shown that the suppression of CD3-subunit expression by TIL was ameliorated in tumors where TGF beta -expression was reduced by inducible TGF beta-specific antisense-RNA, thus, suggesting that lymphocytes may become de-activated upon infiltration of the tumor micro-environment.

Transforming growth factor β in infectious disease: always there for the host and the pathogen

At the portals of pathogen entry, there are pools of the latent form of a potent cytokine, transforming growth factor beta (TGF-beta). Many infections activate these pools and stimulate further TGF-beta expression. As well as potent immunomodulation, activated TGF-beta might have important effects on pathogen entry, replication, persistence, latency and oncogenesis.

Preferential binding of Chlamydia trachomatis to subsets of human lymphocytes and induction of interleukin-6 and interferon-gamma

The interactions between Chlamydia trachumatis and human blood mononuclear leukocytes were studied using flow cytometry. Immunofluorescence, electron microscopy and cytokine assays. Under serum‐free conditions, elementary bodies (EB) of C. trachomatis were found to bind to human T lymphocytes as well as to B cells and monocytes/macrophages (Mφ). For all cell types the binding was saturable. rapid, temperature‐independent and independent of the chlamydia‐specific serological status of the donor. Similar proportions of T and B cells bound EB at similar levels. In the T cell population, proportionally less CD8+cells bound EB. Whereas Mφ phagocytosed and destroyed the bound micro‐organisms for lymphocytes, the Chlamydia remained at the surface, adherent to morphologically featureless membrane areas and showed no evidence of uptake even after long periods at 37°C. Host molecules modulated these basic binding patterns: a heat‐stable serum factor inhibited EB binding to T cells and a heat‐labile serum factor enhanced binding to B cells. Stimulation with C. trachomatis EB rapidly elicited cytokine production by lymphocytes including interleukin‐6 from B cells and interferon‐gamma (IFN‐γ) from T and/or nonT/nonB cells. The responses were irrespective of the serological status of the donor. The findings suggest that C. trachomati‐leucocyte interactions may differ from the interactions of other bacteria and human leucocytes. The possible relationship between leucocyte‐binding, cytokine induction, and the pathognomonic development of lymphoid follicles during mucosal C. trachomatis infections is discussed.

Potential for cytokine therapy of malignant mesothelioma

*Transplantation Biology Unit, K Floor, Queensland Institute of Medical Research, 300 Herston Road, Herston, Queensland 4029, Australia; tCell Biology Research Unit, Z Block Fremantle Hospital, Alma Street, Fremantle, Western Australia 6160, Australia, #Department of Respiratory Medicine, B Block, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia SDepartment of Medicine, University of Western Australia, G Block, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia, and