Klaus T. Jensen

Cationic Liposomes Containing Mycobacterial Lipids: a New Powerful Th1 Adjuvant System

ABSTRACT The immunostimulation provided by the mycobacterial cell wall has been exploited for many decades, e.g., in Freunds complete adjuvant. Recently, the underlying mechanism behind this adjuvant activity, including Toll receptor signaling, has begun to be unraveled, confirming the potential of mycobacterial constituents to act as adjuvants. In this study, the immunostimulatory properties of a Mycobacterium bovis BCG lipid extract were tested for their adjuvant activity. Administration of the lipids in dimethyl dioctadecyl ammonium bromide-based cationic liposomes induced a powerful Th1 response characterized by markedly elevated antigen-specific immunoglobulin G2a (IgG2a) isotype antibodies and substantial production of gamma interferon. The adjuvant formulation (designated mycosomes) elicited high levels of gamma interferon both in C57BL/6 as well as in Th2-prone BALB/c mice. Furthermore, the mycosomes induced immune responses to protein antigens from several sources including Mycobacterium tuberculosis, Chlamydia muridarum, and tetanus toxoid. In a tuberculosis challenge model, the mycosomes combined with the Ag85B-ESAT-6 fusion protein were demonstrated to have a unique ability to maintain sustained immunological memory at a level superior to live BCG.

Inhibition of miR-9 de-represses HuR and DICER1 and impairs Hodgkin lymphoma tumour outgrowth in vivo

MicroRNAs are important regulators of gene expression in normal development and disease. miR-9 is overexpressed in several cancer forms, including brain tumours, hepatocellular carcinomas, breast cancer and Hodgkin lymphoma (HL). Here we demonstrated a relevance for miR-9 in HL pathogenesis and identified two new targets Dicer1 and HuR. HL is characterized by a massive infiltration of immune cells and fibroblasts in the tumour, whereas malignant cells represent only 1% of the tumour mass. These infiltrates provide important survival and growth signals to the tumour cells, and several lines of evidence indicate that they are essential for the persistence of HL. We show that inhibition of miR-9 leads to derepression of DICER and HuR, which in turn results in a decrease in cytokine production by HL cells followed by an impaired ability to attract normal inflammatory cells. Finally, inhibition of miR-9 by a systemically delivered antimiR-9 in a xenograft model of HL increases the protein levels of HuR and DICER1 and results in decreased tumour outgrowth, confirming that miR-9 actively participates in HL pathogenesis and points to miR-9 as a potential therapeutic target.

Liposome Delivery of Chlamydia muridarum Major Outer Membrane Protein Primes a Th1 Response That Protects against Genital Chlamydial Infection in a Mouse Model

BACKGROUND Immunity to chlamydia is thought to rely on interferon (IFN)-gamma-secreting T helper cells type 1 (Th1) with an additional effect of secreted antibodies. A need for Th1-polarizing adjuvants in experimental chlamydia vaccines has been demonstrated, and antigen conformation has also been reported as being important for raising protective immunity. METHODS C57BL/6 mice vaccinated with native refolded Chlamydia muridarum major outer membrane protein (MOMP) adjuvanted with either Th1-promoting cationic adjuvant formulation 1 (CAF01) or T helper cells type 2-promoting aluminum hydroxide (alum) received a genital inoculation of 1.5 x 10(5) inclusion-forming units of C. muridarum. The role played by CD4(+) T cells in MOMP/CAF01-raised immunity was investigated by depleting CD4(+) T cells in vaccinated mice, and antigen conformation dependence was evaluated by vaccination with recombinant MOMP. RESULTS Mice vaccinated with MOMP/alum displayed a strong anti-MOMP humoral response with high IgG1 titers, low levels of IFN-gamma and tumor necrosis factor (TNF)-alpha, and only a slight reduction in chlamydial load. Mice vaccinated with MOMP/CAF01 displayed high titers of IgG2b, IFN-gamma, and TNF-alpha and a profoundly reduced vaginal chlamydial load, compared with control mice. The protection was CD4(+) T cell dependent and was not dependent on MOMP conformation. CONCLUSION CAF01 adjuvant facilitates a protective anti-MOMP CD4(+) T cell response independent of MOMP conformation.

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia‐inducible factor 1 (HIF‐1). HIF‐1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF‐1 and mediates the hypoxic repression of a set of nuclear‐encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear‐encoded mitochondrial genes where it directly antagonizes c‐Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short‐hairpin RNA (shRNA)‐expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.

Prdm5 Regulates Collagen Gene Transcription by Association with RNA Polymerase II in Developing Bone

PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix.

Antigenic Profiling of a Chlamydia trachomatis Gene-Expression Library

The obligate intracellular bacterium Chlamydia trachomatis is the causative agent of sexually transmitted chlamydia infections. A panel of 116 recombinant C. trachomatis proteins was evaluated comparatively to characterize both cell-mediated and humoral immune responses in patients with confirmed C. trachomatis genital infection. The antigens identified were categorized as being recognized exclusively by T cells (CT004, CT043, CT184, CT509, and CT611), B cells (CT082, CT089, CT322, CT396, and CT681), or both T cells and B cells (CT110 and CT443). This grouping of C. trachomatis antigens was correlated to their predicted cellular localization. The comparative evaluation presented here indicates that T cell antigens are located in all bacterial compartments, whereas antibody targets are mainly localized to the outer membrane (P = .0013). Overall, we have identified 5 T cell antigens, 5 B cell antigens, and 2 T/B cell antigens that are potential components for a future chlamydia vaccine.

Chromatin-modifying proteins in cancer.

Chromatin‐modifying proteins mold the genome into areas that are accessible for transcriptional activity and areas that are transcriptionally silent. This epigenetic gene regulation allows for different transcriptional programs to be conducted in different cell types at different timepoints–despite the fact that all cells in the organism contain the same genetic information. A large amount of data gathered over the last decades has demonstrated that deregulation of chromatin‐modifying proteins is etiologically involved in the development and progression of cancer. Here we discuss how epigenetic alterations influence cancer development and review known cancer‐associated alterations in chromatin‐modifying proteins.

Loss of miR-10a activates lpo and collaborates with activated Wnt signaling in inducing intestinal neoplasia in female mice.

miRNAs are small regulatory RNAs that, due to their considerable potential to target a wide range of mRNAs, are implicated in essentially all biological process, including cancer. miR-10a is particularly interesting considering its conserved location in the Hox cluster of developmental regulators. A role for this microRNA has been described in developmental regulation as well as for various cancers. However, previous miR-10a studies are exclusively based on transient knockdowns of this miRNA and to extensively study miR-10a loss we have generated a miR-10a knock out mouse. Here we show that, in the Apcmin mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10+/+ and male controls. We further found that Lpo is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using in vitro assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of Lpo, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of miR-10a knockout mice. Lpo has previously been shown to have the capacity to oxidize estrogens into potent depurinating mutagens, creating an instable genomic environment that can cause initiation of cancer. Therefore, we postulate that Lpo upregulation in the intestinal epithelium of miR-10a deficient mice together with the predominant abundance of estrogens in female animals mainly accounts for the sex-related cancer phenotype we observed. This suggests that miR-10a could be used as a potent diagnostic marker for discovering groups of women that are at high risk of developing colorectal carcinoma, which today is one of the leading causes of cancer-related deaths.

Identification of CT521 as a frequent target of Th1 cells in patients with urogenital Chlamydia trachomatis infection.

BACKGROUND The human immune response to a Chlamydia trachomatis serovar D lysate was investigated in patients with urogenital C. trachomatis infection, to identify novel T cell targets. METHODS A C. trachomatis lysate was fractionated on the basis of molecular mass, and each fraction was used to stimulate peripheral-blood mononuclear cells from patients with C. trachomatis infection. In frequently recognized fractions, proteins were identified by mass spectrometry, recombinantly expressed, and tested for T cell recognition. RESULTS T cell recognition of the fractions was highly heterogeneous in patients with C. trachomatis infection (n=16). Four patients exhibited responses that were strongly targeted to antigens of 16-20-kDa molecular mass. Three proteins were identified in this fraction: CT043, CT511, and CT521. The T cell response to the individual recombinant proteins were investigated, and CT521 was found to induce the highest level of interferon (IFN)- gamma. The recognition of CT521 was investigated in a larger study population (n=41), and a positive IFN-gamma response was measured in 83% of the patients. Several T cell epitopes were identified in CT521; in particular, peptide 5 in the central part of the protein was frequently recognized by T cells (63%). CONCLUSION We have identified a novel C. trachomatis antigen, CT521, that is frequently recognized in patients with urogenital C. trachomatis infection.

Loss of PRDM11 promotes MYC-driven lymphomagenesis

The PR-domain (PRDM) family of genes encodes transcriptional regulators, several of which are deregulated in cancer. By using a functional screening approach, we sought to identify novel tumor suppressors among the PRDMs. Here we demonstrate oncogenic collaboration between depletion of the previously uncharacterized PR-domain family member Prdm11 and overexpression of MYC. Overexpression of PRDM11 inhibits proliferation and induces apoptosis. Prdm11 knockout mice are viable, and loss of Prdm11 accelerates MYC-driven lymphomagenesis in the Eµ-Myc mouse model. Moreover, we show that patients with PRDM11-deficient diffuse large B-cell lymphomas (DLBCLs) have poorer overall survival and belong to the nongerminal center B-cell-like subtype. Mechanistically, genome-wide mapping of PRDM11 binding sites coupled with transcriptome sequencing in human DLBCL cells evidenced that PRDM11 associates with transcriptional start sites of target genes and regulates important oncogenes such as FOS and JUN. Hence, we characterize PRDM11 as a putative novel tumor suppressor that controls the expression of key oncogenes, and we add new mechanistic insight into B-cell lymphomagenesis.