Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences

BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA

Dynamics of the major histocompatibility complex class I processing and presentation pathway in the course of malaria parasite development in human hepatocytes: implications for vaccine development.

Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver

Effect of <i>P. falciparum</i> 3D7 GFP infection on surface MHC class I expression.

<p>Primary human hepatocytes were infected with <i>P. falciparum</i> 3D7 GFP sporozoites and surface MHC class I expression was assessed in GFP+ and GFP- cell populations at days 5 and 8 post-infection using immunostaining followed by flow cytometry (for details see Materials and Methods). (A) Dot plots (gray) show side scatter characteristics of GFP- cells (uninfected), while the contour plots (black) show GFP+ (infected) populations at day 5 and day 8 post-infection. Filled histograms represent staining with an isotype control antibody; solid lines, staining with a MHC class I-specific antibody in both populations at day 5 and day 8. Data are representative of two independent experiments. (B) Modulation of MHC class I in <i>P. falciparum</i> 3D7 GFP infected primary hepatocytes on day 5 post-infection by recombinant IFNγ and TNFα supplied at day 3 post-infection. Dot plots (GFP-) represent populations of uninfected cells, while contour plots show infected (GFP+) cells. Numbers are MFI of specific fluorescence obtained following staining with an APC-conjugated isotype control or MHC class I-specific antibody. (C) Expression of MHC class I in <i>P. falciparum</i> 3D7 GFP infected primary hepatocytes on day 5 post-infection in response to AS (10% v/v) supplied on day 3. Filled histograms show staining obtained with an isotype control antibody, solid lines - staining obtained with an MHC class I specific antibody. Numbers represent MFI values of cell populations stained for MHC class I expression.</p

Surface MHC class I expression on infected hepatocytes at the early stages of parasite development.

<p>HC-04 cells (A, C, D) or primary freshly isolated human hepatocytes (B) were infected with <i>P. berghei</i> ANKA GFP and surface MHC class I was assessed by flow cytometry in uninfected or GFP+ and GFP- cell populations at 24 and 48 hrs post-infection. The dot plots (A and B) show the proportion of infected (GFP+) cells at 24 hrs post-infection. (A) One representative experiment performed at 24 hrs post-infection. Filled histograms - staining with the isotype control antibody; unfilled - staining with the MHC class I-specific antibody. The numbers are mean fluorescence intensities (MFIs) of MHC class I staining. (B) MHC class I expression on the surface of GFP+, GFP- and uninfected primary human hepatocytes. Filled histograms, staining with the isotype control antibody; solid lines, staining with the MHC class I-specific antibody. (C) Parasite replication does not affect <i>de </i><i>novo</i> formation and transport of MHC class I complexes to the cell surface. HC-04 cells were subjected to treatment with a low pH buffer at 24 hrs post-infection to dissociate surface MHC class I molecules (time zero) and surface MHC class I expression was determined after the indicated periods of time post treatment. Overlay of contour plots for GFP+ and GFP- cells stained with the isotype control or MHC class I-specific antibody at each time point. Numbers indicate MFI in each population. One of three reproducible experiments. (D) MHC class I expression in GFP+ (filled histogram) and GFP- (unfilled histogram) populations 48 hrs post-infection with <i>P. berghei</i>. The arrow within the plot marks the small population of MHC class I negative cells. The bar-graphs show the means ± SD of MFI values obtained in 6 (for 48 hrs) and 9 (for 24 hrs) separate experiments. White and black bars represent staining with the isotype control or MHC class I-specific antibody, respectively. NS, not significant; <i>p</i>** = 0.03.</p

Identification, isolation and MHC class I expression on merosomes.

<p>MHC class I expression in GFP+/SSC^low population was assessed by flow cytometry in unsorted (A) and sorted (B) <i>P. berghei</i> ANKA GFP infected HC-04 cultures. (A) The dot plot graph demonstrates identification of GFP-, GFP+/SSC^high and GFP+/SSC^low populations. The histograms demonstrate MHC class I expression (solid line) and staining with an isotype control antibody (filled histograms). The numbers indicate values of relevant MFI. (B) GFP+/PI- HC-04 cells (R1) were separated into GFP+/SSC^high (R2) and GFP+/SSC^low (R3) by cell sorting and MHC class I expression was monitored in each population separately. In parallel, cells were observed by immunofluorescence. (C) Cytospin preparations of sorted cells or subcellular structured from gates R2 (a,b,c) and R3 (d,e,f,g) as shown on panel B were fixed and mounted with DAPI-containing mounting media. Presence of host and parasite-derived DNA was monitored using a Nikon90i microscope at 40X (a) and 100X (b-g). Scale bars indicate 20 µm (a) and 10 µm (b-g).</p

Flow cytometry-based detection of HC-04 cells infected with <i>P. berghei</i> ANKA GFP.

<p>(A) HC-04 cells infected with <i>P. berghei</i> ANKA GFP sporozoites were detected by flow cytometry based on GFP expression. (B) GFP+ cells were isolated at 24 hrs post-infection by flow cytometry-based cell sorting and stained with DAPI (a, b). Parasitophorous vacuoles (PVs) are indicated by arrows. (c) GFP+ cells isolated at 48 hrs post-infection and stained with DAPI contain large PVs and some cells (d) resemble merosome-like structures. (C) mRNA expression of <i>P. berghei</i> ANKA GFP genes <i>18S, CSP</i> and <i>HSP70</i> was analyzed by RT-PCR in GFP+ and GFP- cells purified by sorting from infected cultures. Samples were collected at 24 and 48 hrs post-infection. HC-04 cells from non-infected cultures were used as a negative control. Data from two independent experiments are shown in the figure.</p

Activation of CTLs exposed to human hepatocytes infected with <i>P. berghei</i> ANKA GFP.

<p>(A) Activation of GLC peptide specific CTLs was done using uninfected, GFP- and GFP+ HC-04 cells as stimulators as described in Materials and Methods. One representative experiment of 3 performed is shown. Numbers indicate percentages of positive cells for each activation marker indicated in the figure. (B) Cytokine release from HLA-A2 specific allogeneic T cells activated on infected and uninfected HC-04 cells is described in Materials and Methods. Summary of 3 independent experiments. HLA-A2 positive LCL was used as a control of T cell activation. NS, not significant.</p

Circumsporozoite protein does not affect basal or inducible expression of MHC class I.

<p>(A) Expression of the full length (CSPf.l.) or mature “short” (CSPsh) form of the <i>P. falciparum</i> 3D7 CSP protein was detected by western blotting 24 hrs after transfection of HC-04 cells. (B) Cellular distribution of full length CSP 24 hrs post-transfection was visualized with a CSP-specific antibody (green) and immunofluorescence microscopy. Nuclei were stained with DAPI (blue). Arrows indicate transfected cells. (C) Real-time PCR analysis of MHC class I heavy chain, β2-microglobulin and TAP1 gene expression in cells transiently expressing of <i>P. falciparum</i> CSP. HC-04 cells transfected with the control vector plasmid or plasmid encoding either full length or short CSP were treated with AS (10% v/v) 4 hrs post transfection or left untreated. Transfected cells were isolated 24 hrs later using surface expression of mouse CD8α as a marker. Mean ± SD of the assay triplicates. All <i>p</i>* <0.0002 and indicated differences between control and AS-treated samples. (D) Percentages of cells transiently expressing CSP were identified by CD8α co-expression (upper panels) and MHC class I was assessed by flow cytometry (lower panel, light gray histograms - isotype control, dark gray histogram - MHC class I specific antibody). A proportion of cells exposed to either AS (10% v/v) or a mixture of recombinant TNFα and IFNγ at 24 hrs post transfection was further assessed for MHC class I expression at 48 hrs (light gray histograms - isotype control antibody, dark gray histogram - MHC class I in untreated cultures, solid line histogram – cultures treated with recombinant cytokines, dotted line histogram – cultures treated with AS). Data from one representative experiment. (E) The means ± SD of MFI for specific MHC class I staining obtained in 3 independent experiments. All <i>p</i>* <0.0001 and indicated differences between control and treated samples. (F) Expression of MHC class I heavy chain (HC) in total cell lysates of HC-04 cells transfected with CSP-expressing plasmids was assessed by western blot. Treatment with AS was done as described for D.</p

MHC class I pathway gene expression in hepatocytes infected with <i>P. berghei</i> ANKA GFP.

<p>GFP+ and GFP- HC-04 cells were isolated by FACS sorting at 24 and 48 hrs post-infection from the same parasite<i>-</i>infected cell cultures. Uninfected cells were also subjected to sorting prior to mRNA isolation. Real time PCR analysis of mRNA expression was done for the 15 genes indicated. Each assay was performed in triplicate. Data are shown as expression units relative to GAPDH expression levels arbitrarily taken as 1 and represent the mean ± SD from 4 to 9 independent infection experiments and cell sorting procedures. NS, not significant.</p

Kinetic analysis of AS-mediated effects on MHC class I expression in infected hepatocytes.

<p>HC-04 cultures infected with <i>P. berghei</i> ANKA GFP were exposed to activated supernatant (AS, 10% v/v) at the indicated time points post-infection and surface MHC class I staining was performed at 48 hrs post-infection. (A) Dot plots (GFP-) and contour plots (GFP+) represent MHC class staining with an APC-labeled MHC class I specific antibody or relevant isotype control antibody on infected and uninfected hepatocytes, respectively. One representative of 3 experiments is shown. The numbers indicate MFI values of MHC class I specific staining for respective conditions and populations. The dotted line boxes mark the observed area of events distribution in untreated cultures. (B) Summary of 3 experiments performed as described in A. The means ± SD of MFI values for surface MHC class I staining are shown. All <i>p</i> values are ≤0.01 and indicate differences between control and treated uninfected(*), GFP negative (**) and GFP positive (***) cells.</p