Frans A. Prins

Epidermal Growth Factor-Like Repeats Mediate Lateral and Reciprocal Interactions of Ep-CAM Molecules in Homophilic Adhesions

ABSTRACT Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca2+-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via α-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.

Human Leukocyte Antigen Class I, MHC Class I Chain-Related Molecule A, and CD8+/Regulatory T-Cell Ratio: Which Variable Determines Survival of Cervical Cancer Patients?

Purpose: To investigate the effect of intraepithelial tumor-infiltrating lymphocytes (ieTIL) and their ligands expressed by cervical tumor cells on the outcome of cervical cancer patients. Experimental Design: The prognostic value of ieTILs was investigated in 115 cases of cervical cancer. T-cell subsets, CD57+ cells, and regulatory T cells (Treg) were enumerated. The associations of these different ieTIL subtypes with human leukocyte antigen (HLA) class I and MHC class I chain-related molecule A (MICA) expression were determined in relation to clinical variables and patient survival. Results: Survival analysis showed that a high number of intraepithelial Treg (FoxP3+), a low CD8+/regulatory T-cell ratio, and a weak HLA-A expression were all associated with worse survival (P = 0.034, 0.025, and 0.033, respectively, log-rank test). Further stratification of patient groups based on HLA-A-MICA expression and HLA-A-MICA-CD8+/Treg ratio revealed an even poorer survival (P = 0.005). In a multivariate Cox analysis, low CD8+/Treg ratio (P = 0.047), weak HLA-A-MICA expression (P = 0.003), and weak HLA-A-MICA expression combined with low CD8+/Treg ratio (P = 0.002) were all found to be independent unfavorable prognostic predictors in cervical carcinoma (hazard ratios, 2.7, 4.0, and 4.9, respectively). Conclusion: Weak HLA-A-MICA expression combined with low CD8+/Treg ratio reveals a patient group with the poorest survival in cervical cancer. As a single variable, low CD8+/Treg ratio was a significant independent unfavorable prognostic factor.

Aberrant Chemokine Receptor Expression and Chemokine Production by Langerhans Cells Underlies the Pathogenesis of Langerhans Cell Histiocytosis

Langerhans cell histiocytosis (LCH) is characterized by a clonal proliferation and retention of cells with a Langerhans cell (LC)-like phenotype at various sites within the body. The present study set out to elucidate whether aberrant expression of chemokine receptors or dysregulation of chemokine production in LCH lesions could explain abnormal retention of these cells. Immunohistochemical analysis on 13 LCH biopsies of bone, skin, and lymph node all expressed the immature dendritic cell (DC) marker CCR6 on the lesional LCs and absence of the mature DC marker CCR7. Furthermore, regardless of the tissue site, LCH lesions markedly overexpressed CCL20/MIP-3α, the ligand for CCR6. The lesional LCs appeared to be the source of this CCL20/MIP-3α production as well as other inflammatory chemokines such as CCL5/RANTES and CXCL11/I-TAC. These may explain the recruitment of eosinophils and CD4+CD45RO+ T cells commonly found in LCH lesions. The findings of this study emphasize that, despite abundant TNF-α, lesional LCs remain in an immature state and are induced to produce chemokines, which via autocrine and paracrine mechanisms cause not only the retention of the lesional LCs but also the recruitment and retention of other lesional cells. We postulate that the lesional LCs themselves control the persistence and progression of LCH.

Distinct defects in collagen microarchitecture underlie vessel-wall failure in advanced abdominal aneurysms and aneurysms in Marfan syndrome

An aneurysm of the aorta is a common pathology characterized by segmental weakening of the artery. Although it is generally accepted that the vessel-wall weakening is caused by an impaired collagen metabolism, a clear association has been demonstrated only for rare syndromes such as the vascular type Ehlers–Danlos syndrome. Here we show that vessel-wall failure in growing aneurysms of patients who have aortic abdominal aneurysm (AAA) or Marfan syndrome is not related to a collagen defect at the molecular level. On the contrary our findings indicate similar (Marfan) or even higher collagen concentrations (AAA) and increased collagen cross-linking in the aneurysms. Using 3D confocal imaging we show that the two conditions are associated with profound defects in collagen microarchitecture. Reconstructions of normal vessel wall show that adventitial collagen fibers are organized in a loose braiding of collagen ribbons. These ribbons encage the vessel, allowing the vessel to dilate easily but preventing overstretching. AAA and aneurysms in Marfan syndrome show dramatically altered collagen architectures with loss of the collagen knitting. Evaluations of the functional characteristics by atomic force microscopy showed that the wall has lost its ability to stretch easily and revealed a second defect: although vascular collagen in normal aortic wall behaves as a coherent network, in AAA and Marfan tissues it does not. As result, mechanical forces loaded on individual fibers are not distributed over the tissue. These studies demonstrate that the mechanical properties of tissue are strongly influenced by collagen microarchitecture and that perturbations in the collagen networks may lead to mechanical failure.

SDHD mutations in head and neck paragangliomas result in destabilization of complex II in the mitochondrial respiratory chain with loss of enzymatic activity and abnormal mitochondrial morphology

Hereditary head and neck paragangliomas are tumours associated with the autonomic nervous system. Recently, mutations in genes coding for subunits of mitochondrial complex II, succinate‐ubiquinone‐oxidoreductase (SDHB, SDHC, and SDHD), have been identified in the majority of hereditary tumours and a number of isolated cases. In addition, a fourth locus, PGL2, has been mapped to chromosome 11q13 in an isolated family. In order to characterize phenotypic effects of these mutations, the present study investigated the immunohistochemical expression of the catalytic subunits of complex II (flavoprotein and iron protein), SDH enzyme activity, and mitochondrial morphology in a series of 22 head and neck paragangliomas. These included 11 SDHD‐, one SDHB‐, two PGL2‐linked tumours, and eight sporadic tumours. In the majority of the tumours (∼90%), the enzyme‐histochemical SDH reaction was negative and immunohistochemistry of catalytic subunits of complex II showed reduced expression of iron protein and enhanced expression of flavoprotein. Ultrastructural examination revealed elevated numbers of tightly packed mitochondria with abnormal morphology in SDHD‐linked and sporadic tumours. Immuno‐electron microscopy showed localization of the flavoprotein on the remnants of the mitochondrial inner membranes, whereas virtually no signal for the iron protein was detected. These results indicate that the function of mitochondrial complex II is compromised in the majority of head and neck paragangliomas. Copyright

Human Decidual Tissue Contains Differentiated CD8+ Effector-Memory T Cells with Unique Properties

During pregnancy, maternal lymphocytes at the fetal–maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4+CD25bright regulatory T cells have been shown to be concentrated in decidual tissue, where they are able to suppress fetus-specific and nonspecific immune responses. Decidual CD8+ T cells are the main candidates to recognize and respond to fetal HLA-C at the fetal–maternal interface, but data on the characteristics of these cells are limited. In this study we examined the decidual and peripheral CD8+ T cell pool for CD45RA, CCR7, CD28, and CD27 expression, using nine-color flow cytometry. Our data demonstrate that decidual CD8+ T cells mainly consist of differentiated CD45RA−CCR7− effector-memory (EM) cells, whereas unprimed CD45RA+CCR7+ naive cells are almost absent. Compared with peripheral blood EM CD8+ T cells, the decidual EM CD8+ T cells display a significantly reduced expression of perforin and granzyme B, which was confirmed by immunohistochemistry of decidual tissue sections. Interestingly, quantitative PCR analysis demonstrates an increased perforin and granzyme B mRNA content in decidual EM CD8+ T cells in comparison with peripheral blood EM CD8+ T cells. The presence of high levels of perforin and granzyme B mRNA in decidual EM T cells suggests that decidual CD8+ T cells pursue alternative means of EM cell differentiation that may include a blockade of perforin and granzyme B mRNA translation into functional perforin and granzyme B proteins. Regulation of decidual CD8+ T cell differentiation may play a crucial role in maternal immune tolerance to the allogeneic fetus.

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma.

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture (‘mosaic’) of normal lining (EXT+/− or EXTwt/wt) and EXT−/− cells in the cartilaginous cap of osteochondromas.

High-resolution multi-parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin-embedded tissues

The accuracy of DNA ploidy measurements of paraffin‐embedded tissues is limited by the lack of resolution and the inability to identify the DNA diploid population unequivocally in bimodal DNA histograms. A multi‐parameter DNA flow cytometric method has been developed that enables the simultaneous detection of neoplastic and stromal cells in samples from dewaxed 50 µm sections or 2 mm diameter punches of archival tissue blocks. The method combines heat pretreatment in sodium citrate buffer and subsequent enzymatic dissociation with a collagenase/dispase mixture. Cells were simultaneously stained for keratin (FITC), vimentin (R‐PE), and DNA (PI) before flow cytometric analysis. The method was applied to 12 paraffin‐embedded cervical carcinomas and four colorectal carcinomas. In all cervical cancers, distinct keratin‐positive and vimentin‐positive cell populations were observed. While the exclusive vimentin‐positive cell fractions always yielded unimodal DNA content distributions, bimodal distributions were observed for the keratin‐positive cell fractions in nine cervical carcinomas, whereas one cervical carcinoma showed three distinct G0G1 populations. Coefficients of variation of the G0G1 peaks ranged from 1.70% to 4.79%. Average background, aggregate, and debris values were 14.7% (vimentin‐positive fraction) and 33.8% (keratin‐positive fraction). Flow sorting confirmed that the exclusively vimentin‐positive cell fractions represent different normal stromal and infiltrate cells that can serve as an internal ploidy reference enabling discrimination between DNA hypo‐diploid and DNA hyper‐diploid tumour cell subpopulations. The neoplastic origin of the keratin–vimentin co‐expressing cells from two cervical carcinomas was confirmed by genotyping of flow‐sorted samples revealing loss of heterozygosity (LOH) of 6p. This improved method obviates the need for fresh/frozen tumour tissue for high‐resolution DNA ploidy measurements and enables the isolation of highly purified tumour subpopulations for subsequent genotyping. Copyright

Ultrastructural evidence of early non-fibrillar Aβ42 in the capillary basement membrane of patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type

Abstract The C-terminal profile and ultrastructure of small and presumably early capillary amyloid β protein (Aβ) deposits were investigated in four patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type. The C terminus of the 40 (Aβ40) or the 42 (Aβ42) amino acid form of Aβ was gold labeled in serial, ultrathin sections on glass slides for reflection contrast microscopy and on grids for electron microscopy. In all studied subjects, reflection contrast microscopy revealed capillaries with focal Aβ42 immunolabeling in the absence of Aβ40 labeling. In the adjacent electron microscopic section, Aβ42 labeling was confined to the capillary basement membrane. The majority of Aβ42+40– deposits showed no amyloid fibrils. Aβ42+40– deposits were sometimes observed in an unremarkable basement membrane but usually showed increased electron density and reticular structures. A small subset of Aβ42+40– deposits with basement membrane changes showed few amyloid fibrils. Aβ42+40+ capillary deposits always showed definite fibrils and were larger than Aβ42+40– capillary deposits. The present findings suggest that in capillaries the accumulation and subsequent polymerization of Aβ42, possibly in conjunction with basement membrane changes, precedes the definite fibril formation with Aβ40.

Sdhd and Sdhd/H19 Knockout Mice Do Not Develop Paraganglioma or Pheochromocytoma

Background Mitochondrial succinate dehydrogenase (SDH) is a component of both the tricarboxylic acid cycle and the electron transport chain. Mutations of SDHD, the first protein of intermediary metabolism shown to be involved in tumorigenesis, lead to the human tumors paraganglioma (PGL) and pheochromocytoma (PC). SDHD is remarkable in showing an ‘imprinted’ tumor suppressor phenotype. Mutations of SDHD show a very high penetrance in man and we postulated that knockout of Sdhd would lead to the development of PGL/PC, probably in aged mice. Methodology/Principal Findings We generated a conventional knockout of Sdhd in the mouse, removing the entire third exon. We also crossed this mouse with a knockout of H19, a postulated imprinted modifier gene of Sdhd tumorigenesis, to evaluate if loss of these genes together would lead to the initiation or enhancement of tumor development. Homozygous knockout of Sdhd results in embryonic lethality. No paraganglioma or other tumor development was seen in Sdhd KO mice followed for their entire lifespan, in sharp contrast to the highly penetrant phenotype in humans. Heterozygous Sdhd KO mice did not show hyperplasia of paraganglioma-related tissues such as the carotid body or of the adrenal medulla, or any genotype-related pathology, with similar body and organ weights to wildtype mice. A cohort of Sdhd/H19 KO mice developed several cases of profound cardiac hypertrophy, but showed no evidence of PGL/PC. Conclusions Knockout of Sdhd in the mouse does not result in a disease phenotype. H19 may not be an initiator of PGL/PC tumorigenesis.