Double in situ hybridization in combination with digital image analysis: A new approach to study interphase chromosome topography

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far

SOX17 expression and its down-regulation by promoter methylation in cervical adenocarcinoma in situ and adenocarcinoma

Aims: SOX17 expression has not been studied in glandular lesions of the uterine cervix like adenocarcinoma in situ (AIS) and invasive adenocarcinomas (AdC), whereas SOX17 promoter CpG island methylation has been reported. Therefore, the aim of this study was to relate the topographical distribution of SOX17 expression and SOX17 methylation status to each other, and to SOX2 expression, human papillomavirus (HPV) type, and physical status of the virus. Methods and results: Immunohistochemistry was used in 45 cases to assess expression of SOX17 and SOX2. SOX17 promoter methylation was determined in 25 cases by means of bisulphite conversion and methylation-specific polymerase chain reaction. SOX17 and SOX2 showed a mutually exclusive expression pattern in normal epithelium, with a sharp delineation in the squamocolumnar junction. SOX17 was found in endocervical columnar and reserve cells, whereas SOX2 was exclusively found in squamous epithelium. In both glandular lesions and cases with coexisting glandular and squamous intraepithelial components, a complex combination of SOX17 and SOX2 expression patterns was seen and mutually exclusive expression was lost. Frequently, gain of expression of SOX2 was found and expression of SOX17 was lost. Methylation of the CpG island in the SOX17 promoter was shown to be strongly associated with loss of expression of SOX17 (P = 0.0016). Conclusions: In this study, we show for the first time a direct correlation between the topographical distribution of SOX17 expression and the methylation status of its gene promoter. This explains the heterogeneity of SOX17 expression in the glandular lesions of the cervix. No correlation was found between HPV type and physical status of the virus on the one hand and methylation status on the other

Switches of SOX17 and SOX2 expression in the development of squamous metaplasia and squamous intraepithelial lesions of the uterine cervix

Aims: The dynamics and topographical distribution of SOX17 and SOX2 expression was studied in the transformation zone (TZ) of the uterine cervix. This TZ is a dynamic area where switches from glandular into squamous epithelium can be recognized, new squamocolumnar junctions are formed, and premalignant lesions originate. SOX17 and SOX2 show mutually exclusive expression patterns in the normal uterine cervix, with SOX2 being exclusively found in squamous epithelium, while SOX17 is detected in endocervical columnar cells and reserve cells. Methods and Results: Normal cervices and squamous intraepithelial lesions (SIL) were studied with immunohistochemistry, methylation of SOX17, human papilloma virus (HPV) genotyping, and in situ hybridization. In the TZ squamous metaplasia originating from these reserve cells can still show SOX17 expression, while also remnants of SOX17-positive immature metaplasia can be recognized in the normal squamous epithelium. SOX17 expression is gradually lost during maturation, resulting in the exclusive expression of SOX2 in the majority of (SIL). This loss of SOX17 expression is independent of methylation of the CpG island in its promotor region. HPV can be detected in SOX17-positive immature metaplastic regions in the immediate vicinity of SOX2-positive SIL, suggesting that switches in SOX17 and 2 expression can occur upon HPV infection. Conclusions: This switch in expression, and the strong association between the distribution of reserve cells and squamous areas within the columnar epithelium in the TZ, suggests that reserve cell proliferations, next to basal cells in the squamous epithelium, are potential targets for the formation of squamous lesions upon viral infection

Mesenchymal Stem Cells Induce T-Cell Tolerance and Protect the Preterm Brain after Global Hypoxia-Ischemia

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 106MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE

The Two Faces of Cervical Adenocarcinoma In Situ

In order of frequency, cervical intraepithelial neoplasia (CIN), combined adenocarcinoma in situ (AIS)/CIN lesions, and solitary AIS are the most prevalent premalignant lesions of the uterine cervix. As the morphologic distinction of these subtypes is not always straightforward, we performed an immunophenotyping analysis to establish distinguishing profiles for each of these squamous and glandular progenitor lesions of cervical carcinoma. A series of 26 premalignant cervical lesions, comprising 13 cases of AIS, of which 7 represented solitary AIS and 6 were combined with CIN (combined AIS/CIN), as well as 13 solitary high-grade CIN lesions, were immunophenotypically analyzed using antibodies directed against p16, p63, bcl-2, and cytokeratins (CK) 5, 7, 8, 13, 17, 18, and 19. Adjacent normal epithelia were also investigated. CIN lesions expressed the full panel of antibodies. Combined AIS/CIN lesions also expressed the full complement of markers in both the AIS and CIN compartments. However, the expression of p63, bcl-2, CK5, and CK17 was lower in AIS compared with CIN. The solitary AIS lesions could be subdivided into 2 subgroups, 1 expressing the full complement of markers and a second group in which no expression of p63, bcl-2, CK5, and a sporadically CK17 expression was observed. We conclude that 2 phenotypically distinct types of AIS can be identified, that is, AIS with a reserve cell marker phenotype and AIS with an endocervical glandular phenotype. These observations support the view that reserve cells are capable of bidirectional premalignant transformation, that is, into CIN and reserve cell-type AIS, as well as combined AIS/CIN. The endocervical type of AIS is probably a result of the unidirectional transformation of progenitor cells within the glandular cell compartment

Development of the Uterine Cervix and Its Implications for the Pathogenesis of Cervical Cancer

Normal development of the uterine cervix has been widely studied and the origin of both the columnar and squamous epithelia, as well as the molecular basis of their differentiation, has been established. The process of early carcinogenesis in the uterine cervix has also been described extensively, in particular with respect to the role of human papillomavirus (HPV) infection. However, questions remain about the progenitor cell(s) that play(s) a role in normal (embryonic and fetal) development, as well as in the oncogenic processes that take place in the transformation zone of the uterine cervix. This chapter describes the development of the human lower female reproductive tract, in particular the cervical squamocolumnar junction, and its implications for the pathogenesis of cervical cancer

Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer