Expression of mesenchymal stem cell marker CD90 on dermal sheath cells of the anagen hair follicle in canine species

The dermal sheath (DS) of the hair follicle is comprised by fibroblast-like cells and extends along the follicular epithelium, from the bulb up to the infundibulum. From this structure, cells with stem characteristics were isolated: they have a mesenchymal origin and express CD90 protein, a typical marker of mesenchymal stem cells. It is not yet really clear in which region of hair follicle these cells are located but some experimental evidence suggests that dermal stem cells are localized prevalently in the lower part of the anagen hair follicle.As there are no data available regarding DS stem cells in dog species, we carried out a morphological analysis of the hair follicle DS and performed both an immunohistochemical and an immunocytochemical investigation to identify CD90⁺ cells. We immunohistochemically evidenced a clear and abundant positivity to CD90 protein in the DS cells located in the lower part of anagen hair follicle. The positive cells showed a typical fibroblast-like morphology. They were flat and elongated and inserted among bundles of collagen fibres.The whole structure formed a close and continuous sleeve around the anagen hair follicle. Our immunocytochemical study allowed us to localize CD90 protein at the cytoplasmic membrane level.Key words: CD90, mesenchymal stem cells, hair follicle, dog.The hair follicle represents an important stem cell niche in the skin. It contains dermal and epithelial stem populations that display distinct properties and localization. While epithelial stem cells reside in the middle region of the hair follicle outer root sheath (Schneider et al., 2009; Lyle et al., 1998; Cotsarelis et al., 1990), dermal stem cells are located in the dermal sheath (DS) (Jahoda, 2003; Jahoda and Reynolds, 2001).The dermal sheath, or fibrous root sheath, is a layer of dense connective tissue that extends along the hair follicle, from the bulb up to the infundibulum. In the anagen hair follicle, it is comprised of mesenchymal cells located among collagen and elastic fibres.The cells are flat and elongated while collagen fibres form a circular inner layer and a longitudinal outer layer in the lower part of hair follicle (VonTscharner and Suter, 1994; Jahoda et al., 1992). At the base of the hair follicle, the DS is connected to the dermal papilla (Scott et al., 2000). The basement membrane, or glassy membrane, separates the DS from the epithelial component of the hair follicle (Scott et al., 2000).Follicular dermal stem cells have a mesenchymal origin and share many properties common to bone marrow-derived mesenchymal stem cells (MSCs) (Hoogduijn et al., 2006). They express the MSC cell-surface marker CD90, show a high colony forming unit ability and can differentiate into several mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes and myocytes (Hoogduijn et al., 2006; Jahoda et al., 2003). They also express neuroprogenitor markers (Hoogduijn et al., 2006) and, finally, they can repopulate the haematopoietic system (Lako et al., 2002). In the literature, we can find different information about stem cell localization: the whole dermal sheath, the peri-bulbar dermal sheath, the dermal papilla (Hoogduijn et al., 2006, McElwee et al., 2003, Gharzi et al., 2003, Jahoda et al., 2003.)CD90 (Thy-1) is a small GPI-anchored protein localized in the outer leaflet of the cell membrane (Low and Kincade, 1985). This protein is present in a large number of tissues and cells, even if a great species variation has been described (Mansour Haeryfar, 2004; Tokugawa et al., 1997; McKenzle and Fabre, 1981). CD90 plays a role in cell-cell interaction events, including intracellular adhesion and cell recognition during development (Saalbach et al., 2000; Morris, 1985), and is considered an important stem cell marker; for this last reason it is commonly used to identify mesenchymal stem cells in vitro (Kern et al., 2007; Yoshimura et al., 2006; Le Blanc and Ringdén, 2006; Pittenger et al., 1999). Furthermore, it has been identified in other kinds of stem cells such as haematopoietic progenitor cells (Craig et al., 1993) and hepatic progenitor cells in the human fetal liver (Masson et al., 2006).The hair follicle is the focus of increasing interest because it contains well defined stem cell populations that exhibit various developmental properties. We retain that in dogs, as already demonstrated in other species (Hoogduijn et al., 2006; Zhang et al., 2006; Jahoda et al., 2003; Lako et al., 2002), this organ may be a suitable and accessible source for both epithelial and mesenchymal stem cells that may be isolated and in vitro cultured. Since it is possible to take skin samples without injuring the patient, we chose the hair follicle to study and identify stem cells with the future purpose of using them in regenerative medicine.Dogs are affected by several skin diseases and some of them may be related to alterations of somatic stem cells. We retain that the study of hair follicle stem cell biology may improve our knowledge of etiology and pathogenesis of these skin diseases.In previous works we investigated the stem cells in dog hair follicles; we identified the location of putative epithelial stem cells at the isthmus and described the bulge-like region (Pascucci et al., 2006; Mercati et al., 2008). To the authors’ knowledge, there are no data available neither concerning the localization of DS stem cells nor concerning the expression of CD90 in the hair follicle as regards the canine species. Therefore, in this study, we described the morphological characteristics of DS cells and examined the immunohistochemical localization of CD90 protein in dog hair follicles with both light and transmission electron microscopy. The aim of our study is to observe the dermal sheath cells encompassing the hair follicle and to determine where CD90⁺ cells reside. CD90 is one of the main markers used to identify mesenchymal stem cells and it has been observed in stem cells isolated from the dermal sheath of hair follicles (Hoogduijn et al.,2006). For this reason, we suppose that CD90 protein can help us to identify the hair follicle dermal stem compartment in dog.     

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.Key words: versican, dentine matrix, immunohistochemistry, TEM, FEISEM, fluorescence microscope.The human dentine organic matrix is composed by a large complex of macromolecules capable of self-assembly. The dentine matrix is represented predominantly by type I collagen and completed by non collagenous glycoproteins, elastin, hyaluronan and proteoglycans (PGs). While type I collagen is the backbone of the dentine with a predominant structural role, non-collagenous proteins, and in particular PGs, are believed to play fundamental functional roles during odontogenesis, mineralization and homeostasis of dentine.The process of odontogenesis appears to be controlled by a precise sequential expression of a pool of extracellular non-collagenous proteins that induces modifications within the extracellular environment of the predentine leading to the formation of the dentine matrix (Embery et al., 2001). Similarly, dentine mineralization involves a dynamic transition from the unmineralised predentine to the mineralised mature dentine, in which the role of specific regulative mineralisation proteins appears to be pivotal in the precipitation of the minerals and in the formation of apatite crystals (Embery et al., 2001). In particular, PGs has been shown to play crucial role in the mineralisation processes of dentine (Embery et al., 2001; Waddington et al., 2003).PGs are macro-molecules where, at least, one glycosaminoglycan side chain (GAGs) is covalently attached to the protein core of the molecules.Their size and structure can change and can be differentially found intracellulary, on the cell surface, or within the extracellular matrix.The majority of PGs have been identified by their antigenic and structural properties suggesting numerous biological functions (Embery et al., 2001). Biochemical, histochemical and immunohistochemical studies on PGs of dentine and predentine have yielded sufficient information to indicate that the predominant PGs belong to the small leucine-rich interstitial family (SLRP) (Fisher et al., 1983; Yoshiba et al., 1996). They include decorin and biglycan (Waddington et al., 2003; Orsini et al., 2007), which bear one or two chondroitin/dermatan sulphate GAGs, lumican, fibromodulin and osteoadherin that bear keratan sulphate GAGs chains (Iozzo et al., 1997, 1999; Neame et al., 2000). A second pool of PGs belongs to the large aggregation chondroitin/keratan sulphate family named hyaluronan-binding (HA), including aggrecan, versican, brevican and neurocan (Yamauchi et al., 1997).Versican was firstly isolated in chicken mesenchymal tissue, and it has been found to be expressed also in keratinocytes, smooth muscle cells of the vessels, brain and mesangial cells of the kidney. Similar PGs have been found in other connective tissues (Zimmermann et al., 1989; Shinomura et al., 1990; Zimmermann et al., 1994; Landolt et al.,1995) and recent studies have shown that, within the dental tissues, versican has been localised in gingival fibroblasts culture, dental pulp complex (Yamauchi et al., 1997; Bartold et al., 1995; Shibata et al., 2000; Shibata et al., 2002; Robey et al., 1993; Ababneh et al., 1999; Cheng et al., 1999), dentine Waddington et al., 2003), cementum (Ababneh et al., 1999; Cheng et al., 1999) and periodontal ligament (Sato et al., 2002).Within the dentine organic matrix versican can be detected either as fragments or as whole molecule. Waddington et al. (2003) reported that versican is mainly present as its degradation products (fragments), whereas the whole molecule has been isolated by Shibata et al. (1999; 2000) in rat dental pulp tissue.The aim of this study was to localise versican PG in human mature dentine by an immunohistochemical technique using a monoclonal antibody anti-versican (towards the whole molecule) and a polyclonal antibody anti-versican fragments, under high resolution field emission in-lens scanning electron microscope (FEI-SEM), electron transmission microscope (TEM) and fluorescence microscope (FM) and to confirm the morphological findings by a biochemical assay.     

Activation of PKC-ε counteracts maturation and apoptosis of HL-60 myeloid leukemic cells in response to TNF family members

Protein kinase C (PKC)-ε, a component of the serine/threo-nine PKC family, has been shown to influence the survival and differentiation pathways of normal hematopoietic cells. Here, we have modulated the activity of PKC-ε with specific small molecule activator or inhibitor peptides. PKC-ε inhibitor and activator peptides showed modest effects on HL-60 maturation when added alone, but PKC-ε activator peptide significantly counteracted the pro-maturative activity of tumor necrosis factor (TNF)-α towards the monocytic/macrophagic lineage, as evaluated in terms of CD14 surface expression and morphological analyses. Moreover, while PKC-ε inhibitor peptide showed a reproducible increase of TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis, PKC-ε activator peptide potently counteracted the pro-apoptotic activity of TRAIL. Taken together, the anti-maturative and anti-apoptotic activities of PKC-ε envision a potentially important proleukemic role of this PKC family member.Key words: acute myeloid leukemia, surface antigens, HL-60 cells, apoptosis, maturation.Activation of all protein kinase C (PKC) family of serine and threonine isoenzymes is associated with binding to the negatively charged phospholipids, phosphatidylserine, while different PKC isozymes have varying sensitivities to Ca²⁺ and lipid-derived second messengers such as diacylglycerol (Gonelli et al., 2009). Upon activation, PKC isozymes translocate from the soluble to the particulate cell fraction, including cell membrane, nucleus and mitochondria (Gonelli et al., 2009). PKC primary sequence can be broadly separated into two domains: the N-terminal regulatory domain and the conserved C-terminal catalytic domain.The regulatory domain of PKC is composed of the C1 and C2 domains that mediate PKC interactions with second messengers, phospholipids, as well as inter and intramolecular protein-protein interactions. Differences in the order and number of copies of signaling domains, as well as sequence differences that affect binding affinities, result in the distinct activity of each PKC isozyme (Gonelli et al., 2009).In recent years, a series of peptides derived from PKC have been shown to modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Brandman et al., 2007, Souroujon and Mochly-Rosen, 1998). Focusing on PKC-ε isozyme and using a rational approach, one C2-derived peptide that acts as an isozyme-selective activator (Dorn et al., 1999) and another that acts as a selective inhibitor (Johnson et al., 1996) of PKC-ε, have been identified.These findings are particularly interesting since besides being involved in the physiology of normal cardiac (Braun and Mochly-Rosen, 2003, Johnson et al., 1996, Li et al., 2006), hematopoietic (Gobbi et al., 2009, Mirandola et al., 2006, Racke et al., 2001), and neuronal (Borgatti et al., 1996) cell models, mounting experimental evidences have linked altered PKC-ε functions to solid tumor development (Okhrimenko et al., 2005, Gillespie et al., 2005, Lu et al., 2006). Therefore, taking advantage of the recent availability of small molecule peptides able to activate or inhibit specifically PKC-ε by disrupting protein/protein interactions (Dorn et al., 1999, Johnson et al., 1996), which open important therapeutic perspectives, we have investigated the effects of both PKC-ε activator and PKC-ε inhibitor peptides on the maturation and survival of leukemic cells, using as a model system the HL-60 myeloblastic leukemia cell line, which can be induced to undergo terminal differentiation or apoptotic cell death by a variety of chemical and biological agents (Breitman et al., 1980, Zauli et al., 1996).     

Identification of the novel localization of tenascinX in the monkey choroid plexus and comparison with the mouse

Tenascin-X (Tn-X) belongs to the tenascin family of glycoproteins and has been reported to be significantly associated with schizophrenia in a single nucleotide polymorphism analysis in humans. This finding indicates an important role of Tn-X in the central nervous system (CNS). However, details of Tn-X localization are not clear in the primate CNS. Using immunohistochemical techniques, we found novel localizations of Tn-X in the interstitial connective tissue and around blood vessels in the choroid plexus (CP) in macaque monkeys. To verify the reliability of Tn-X localization, we compared the Tn-X localization with the tenascin-C (Tn-C) localization in corresponding regions using neighbouring sections. Localization of Tn-C was not observed in CP. This result indicated consistently restricted localization of Tn-X in CP. Comparative investigations using mouse tissues showed equivalent results. Our observations provide possible insight into specific roles of Tn-X in CP for mammalian CNS function.Key words: tenascin-X, choroid plexus, monkey, mouse, Ehlers-Danlos syndrome, schizophrenia.The tenascins (Tn) are a family of four glyco-protein members – tenascin-C (Tn-C), tenascin-R (Tn-R), tenascin-W (Tn-W) and tenascin-X (Tn-X) – found diversely in the extra-cellular matrix of vertebrate organs (Hsia and Schwarzbauer, 2005; Tucker and Chiquet-Ehrismann, 2009). Important functions of Tn have been investigated in developmental cell adhesion modulation and pathological conditions such as wound healing and tumourigenesis (Adams and Watt, 1993; Hsia and Schwarzbauer, 2005; Tucker and Chiquet-Ehrismann, 2009). Tn-C and Tn-R are prominent in the nervous system and play a role in the development of neurite outgrowth and postnatal synaptic plasticity (Yamaguchi, 2000; Chiquet-Ehrismann and Tucker, 2004; Dityatev and Schachner, 2006). Tn-W is found abundantly in the developing bone and stroma of certain tumours (Chiquet-Ehrismann and Tucker, 2004; Tucker and Chiquet-Ehrismann, 2009). Tn-X is the first tenascin member shown to be clearly associated with the human connective tissue disorder Ehlers–Danlos syndrome (EDS; Burch et al., 1997). Patients with a Tn-X deficiency suffer from skin hyperextensibility, joint hypermobility and poor wound healing ability (Bristow et al., 2005). These symptoms are caused by the occurrence of abnormal irregular collagen fibres. Tn-X plays a role in collagen fibrillogenesis by directly binding to collagen (Mao et al. 2002; Minamitani et al. 2004). Mice with a Tn-X deficiency also showed skin symptoms comparable with those of EDS (Mao et al., 2002).Interestingly, in an analysis of human single nucleotide polymorphisms, Tn-X was reported to be significantly associated with schizophrenia (Wei and Hemmings, 2004; Tochigi et al., 2007). However, thus far, there have been no neuroanatomical reports on the involvement of Tn-X in schizophrenia. In the mammalian central nervous system (CNS), Tn-X mRNA expression has only been shown in the rat meninges of the olfactory bulb (Deckner et al., 2000). Recently, we found novel Tn-X localizations in the adult mouse leptomeninges trabecula in the cerebral cortex and in the connective tissue in the lateral ventricle choroid plexus (CP; Imura and Sato, 2008). Our finding of Tn-X localization in CP, which produces cerebrospinal fluid (CSF), might be a key factor in the investigation of the association between CSF metabolism and enlarged ventricles in schizophrenia. Enlarged ventricles are typical structural abnormalities associated with schizophrenia (Staal et al., 1999). Furthermore, CP secretes biologically active molecules into the CSF for brain development, activity and protection (Strazielle and Ghersi-Egea, 2000; Brown et al., 2004; Thouvenot et al., 2006; Johanson et al., 2008). In these molecules, for instance, there is a brain-derived neurotrophic factor (BDNF), the gene expression level and polymorphism of which have been analysed in relation to the pathogenesis of schizophrenia (Buckley et al., 2007). One study reported that BDNF is able to stimulate Tn-X expression in vitro (Takeda et al., 2005).The validity and limitations of animal models (rodents and monkeys) for use in the study of schizophrenia have been discussed (Tordjman et al., 2007). The authors concluded that monkeys appear to be an interesting social interaction model, more so than rodents, because of their complex well-organized social structure. In addition to differences in social structure, the dopaminergic system of rats and monkeys is quite different (García-Cabezas et al., 2009), and dysfunction of the dopaminergic system is related to schizophrenia (Wang et al. 2008).The CSF outflow system has been studied in some animal models (Kapoor et al., 2008). An anatomical difference in arachnoid granulations has been shown between rodents and monkeys (Krisch, 1988). Arachnoid granulations in monkeys are structurally similar to those in humans (Cooper, 1958; Krisch, 1988). In contrast, arachnoid granulations in rodents are similar to those of cats and dogs (Krisch, 1988). It is possible that Tn-X localization in CP is different between rodents and monkeys.Therefore, details concerning Tn-X localization in monkey CP need to be clarified. In the present study, we compared the immunohistochemistry of Tn-X in monkey CP with that in mouse CP. Subsequently, to verify the reliability of Tn-X localization, we compared it with Tn-C localization in corresponding regions using neighbouring sections.     

Effects of castration on the expression of the NGF and TrkA in the vas deferens and accessory male genital glands of the rat

Nerve Growth Factor (NGF) is a member of the neurotrophin family. Neurotrophins exert their effects by binding to corresponding receptors, which are formed by the tyrosine protein kinases TrkA, TrkB, and TrkC, and the low affinity p75NTR receptor. The role of neurotrophins in the biology of male genital organs is far from clear. In particular, little is known about the influence of sex hormones on the expression of neurotrophins and their receptors. In the present study, using immunohistochemistry and real time RT-PCR, we investigated the expression of NGF and TrkA in the vas deferens and accessory male genital glands in normal and castrated rats.In normal rats, both NGF- and TrkA-immunoreactivities (IR) were localized in the epithelial layer of the vas deferens. NGF-IR was also found in the stroma and epithelium of the vesicular gland and prostate. TrkA-IR was distributed in the epithelial cells of vesicular and prostate glands. The nerves were weakly immunoreactive in all the examined organs. After castration the immunoreactivities increased. Real-time RT-PCR experiments indicated that NGF and TrkA mRNA levels increased significantly after castration. These results suggest that NGF and TrkA are expressed in the internal male genital organs of the rat and that their expression is downregulated by androgen hormones. We hypothesize NGF and TrkA play a role in the processes that regulate the involution of these organs under conditions of androgen deprivation.Key words: androgen hormones, stromal cells, immunohistochemistry, real-time RT-PCR, prostate.Nerve growth factor (NGF) is a member of the neurotrophin family, a family of neurotrophic factors that also includes Brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophins 4/5 (NT4/5). Neurotrophins have essential roles in the survival, development and differentiation of neurons in the central and peripheral nervous systems (Levi-Montalcini, 1987; Ernfors et al., 1994; Snider, 1994; Barbacid, 1995; Huang and Reichart, 2001; Murer et al., 2001). Furthermore, recent data show that neurotrophins are involved in a variety of biological processes in nonneuronal tissues (Yamamoto et al., 1996; Sariola, 2001; Leon et al., 1994; Rosenbaum et al., 1998; Tessarollo, 1998). The biological effects of neurotrophins are mediated by tyrosine kinase receptors encoded by the trk protooncogene family, known as TrkA,TrkB and TrkC (Barbacid, 1995; Lewin and Barde, 1996; Patapoutian and Reichart, 2001). The Trk receptors are specific for their ligands; NGF is the preferred ligand for TrkA, BDNF and NT-4/5 are preferred ligands for TrkB and NT-3 is the preferred ligand for TrkC. In addition, all neurotrophins are recognized by a more widely expressed low-affinity receptor known as panneu-rotrophinreceptor p75NTR, which is a member of the tumor necrosis factor (TNF) receptor family (Teng &Hempstead, 2004).The presence of neurotrophins in the accessory male genital tissues has been well documented. NGF and large quantities of NGF have been found in the vesicular and prostate glands and are related to the rich sympathetic innervation of these organs (Harper et al., 1979, 1982; Harper and Thoenen, 1980; Hofmann and Unsicker, 1982).NGF and its receptors (TrkA, p75NTR) have been immunohistochemically expressed in the reproductive organs of the adult male rats (Li et al., 2005). In the prostate, NGF and NGF precursor have been immunohistochemically localized in the glandular epithelium, suggesting that secretory epithelial cells are the site of production of this factor (Shikata et al., 1984; MacGrogan et al., 1991; Paul et al., 1996). Paracrine neurotrophin synthesis by stromal cells has also been postulated (Pflug et al,. 1995; Dalal and Djakiew, 1997; Weeraratna et al., 2000). High- and low-affinity neurotrophin receptors have been recognized in the nerves and epithelial cells of the prostate (Weeraratna et al., 2000; Graham et al., 1992; MacGrogan et al., 1992; Paul and Habib, 1998; Guate et al., 1999), thus indicating that neurotrophins play a role as growth-regulating factors in this gland.The exact role of neurotrophins in the biology of male genital organs, however, is far from clear. Recently, in the vas deferens and accessory male genital glands of the rat, the expression of the BDNF and its receptors (TrkB and p75NTR) has been reported to be regulated by androgen hormones (Mirabella et al., 2006; Mirabella et al., 2008). In castrated rats, moreover, BDNF has been hypothesized to regulate, via interacting p75NTR, the castration-induced regression of the sympathetic innervation (Mirabella et al., 2006).The present study has, therefore, been undertaken to elucidate the presence and localization of NGF and TrkA in the vas deferens and accessory male genital glands of the rat. In addition, the expression of these proteins and their mRNAs have been determined after castration in order to evaluate whether this neurotrophin and its specific receptor are under the control of androgens.     

Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone-secreting adrenal phaeochromocytoma: review of the literature and report of a case

Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochromocytoma, causing ectopic Cushing’s syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous, 9-cm mass in the right adrenal gland, and [¹¹¹In-DTPA⁰] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region, corresponding to the adrenal mass. The patient underwent laparoscopic surgery and formalin-fixed and paraffin-embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed. Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma, which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR₁, SSTR_2A, SSTR_2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing’s disease was dependent on CRH overproduction by the pheochromocytoma, in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas, and provide further evidence for functional SSTR subtype SSTR_2a in a subgroup of pheochromocytomas, suggesting that these tumours may represent potential target for octreotide treatment.Key words: phaeochromocytoma, neuroendocrine tumours, somatostatin receptors, octreotide, corticotropin releasing hormone.Phaeochromocytomas are tumours derived from the chromaffin cells of the sympathoadrenal system, generally associated with cathecolamine overproduction. They represent a rare condition, occurring in less than 0.2% of patients with hypertension. The diagnosis of sporadic phaeochromocytoma is based on clinical history and features characterized by the triad episodic headache, sweating, and tachycardia, but an increasing number of these tumours are diagnosed in patients without classical symptoms (Pacak et al., 2001). Ectopic Cushing’s syndrome is one of the possible, albeit unusual, expression of pheochromocytoma. Up to date, there are few reports of pheochromocytomas producing adrenocorticotropic hormone (ACTH) and/or ACTH precursors (O’Brien T et al., 1992; Chen et al., 1995; White et al., 2000), and even more limited is the number of cases in which pheochromocytoma secrete corticotropin releasing hormone (CRH) (Eng et al., 1999; Bayraktar et al., 2006).Similar to other neuroendocrine tumours, pheochromocytomas often express somatostatin receptors (SSTR) (De Herder and Hofland, 2004), but data on the specific SSTRs subtypes expressed within the tumours are thus far sparse and conflicting and the real therapeutic effectiveness of somatostatin analogue in these tumours is still debated (Reubi et al., 1992; Kubota et al., 1994; Epelbaum et al., 1995; Hofland et al., 1999; Mundschenk et al., 2003; Unger et al., 2004; Ueberberg et al., 2005; Unger et al., 2007).     

Urocortin-like immunoreactivity in the primary lymphoid organs of the duck (Anas platyrhynchos)

Urocortin (UCN) is a 40 aminoacid peptide which belongs to corticotropin-releasing factor (CRF) family. This family of peptides stimulates the secretion of proopiomelanocortin (POMC)-derived peptides, adrenocorticotropic hormone (ACTH), β-endorphin and melanocyte-stimulating hormone (MSH) in the pituitary gland. In the present study, using Western blotting and immunohistochemistry, the distribution of UCN in the primary lymphoid organs of the duck was investigated at different ages. In the cloacal burse and thymus, Western blot demonstrated the presence of a peptide having a molecular weight compatible with that of the mammalian UCN. In the cloacal burse, immunoreactivity was located in the medullary epithelial cells and in the follicular associated and corticomedullary epithelium. In the thymus, immunoreactivity was located in single epithelial cells. Double labelling immunofluorescence studies showed that UCN immunoreactivity completely colocalised with cytokeratin immunoreactivity in both the thymus and cloacal burse. Statistically significant differences in the percentage of UCN immunoreactivity were observed between different age periods in the cloacal burse. The results suggest that, in birds, urocortin has an important role in regulating the function of the immune system.Key words: cloacal burse, thymus, cytokeratin, medullary reticular epithelial cells, CRFUrocortin (UCN) is a 40-amino acid peptide belonging to the mammalian corticotropin- releasing hormone (CRH) family which was first discovered in the rat midbrain (Vaughan et al., 1995). On the basis of its selective ability to bind CRH-receptor type 2 (CRH-R2), different types of UCN, i.e. UCN1, UCN2 and UCN3, have been identified (Lewis et al., 2001; Reyes et al., 2001). In mammals, UCN has been found in the central nervous (Vaughan et al., 1995), digestive (Muramatsu et al., 2000) and immune systems (Bamberger et al., 1998; Kageyama et al., 1999; Baigent et al., 2000), and in genital organs (Petraglia et al., 1996). UCN has also been found to play a role in regulating some CRH-receptor-mediated effects (Turnbull et al., 1999). While UCN2 and UCN3 selectively bind to CRH-R2, UCN1 binds to both CRH-R1 and CRH-R2 and shows a greater affinity to CRH-R2 than CRH alone (Chalmers et al., 1996). Despite the ability to interact with the same receptors, different functions are attributed to UCN and CRH. CRH is the primary neuroregulator of the vertebrate stress response in so far as it has been shown to be the major hypothalamic releasing factor for pituitary adrenocorticotropic hormone, whereas UCN seems not to be involved in the activation of the hypothalamus- hypophysis-adrenal axis (Turnbull et al., 1999). Conversely, UCN influences the function of the cardiovascular and nervous systems by increasing anxiety, decreasing appetite and influencing behavioral activity (Latchman, 2001). In non-mammalian vertebrates, few data have been reported on the presence and the role of UCN.The molecule, however, may have been conserved during vertebrate evolution, given that it has also been detected in amphibians and birds (Kozicz et al., 2002; Cavani et al., 2003; Boorse et al., 2005; Calle et al., 2005). In amphibians, UCN and CRH receptors have been found in the brain as well as in many other organs and tissues, including the pituitary gland, heart, kidney and alimentary canal (Kozicz et al., 2002; Boorse et al., 2005, 2006); thus suggesting a potential role for diverse actions in tissue maintenance and function. In Xenopus laevis, UCN injected in the third ventricle has been found to suppress food intake (Boorse et al. 2005). Moreover, it has been found to act as a cytoprotective factor in tadpole tail during metamorphosis (Boorse et al. 2006). In birds, UCN-ir has been found in neurons of the pigeon paramedian subgriseal mesencephalon which appear to be part of the brain circuitry involved in sympathetic nervous system-mediated behavioral responses to stress (Cavani et al. 2003; Cunha et al., 2007). Intracerebroventricular administered UCN, moreover, has been reported to decrease food intake in the chicken (Zhang et al., 2001). Up until now, however, no data are available regarding the presence and role of UCN in tissues and organs of birds outside the central nervous system (CNS). Since UCN and its receptors have been reported to be extensively expressed in immune tissues and addressed to play important roles in the regulation of the immune response (Baigent, 2001), the present study has investigated the presence and distribution of UCN in the primary lymphoid organs of the duck by means of Western blotting and immunohistochemistry. In addition, in order to verify if UCN also plays a role in the maturation of bird primary lymphoid organs, UCN expression was evaluated at different age periods.     

Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin

Although several studies have shown that the serum levels of osteoprotegerin (OPG) are significantly elevated in patients affected with atherosclerotic lesions in coronary and peripheral arteries, the cellular source and the role of OPG in the physiopathology of atherosclerosis are not completely defined. Therefore, we aimed to investigate the potential contribution of mesenchymal stem cells in the production/release of OPG. OPG was detectable by immunohistochemistry in aortic and coronary atherosclerotic plaques, within or in proximity of intimal vascular smooth muscle cells (SMC). In addition, bone marrow mesenchymal stem cell (MSC)-derived vascular SMC as well as primary aortic SMC released in the culture supernatant significantly higher levels of OPG with respect to MSC-derived endothelial cells (EC) or primary aortic EC. On the other hand, in vitro exposure to full-length human recombinant OPG significantly increased the proliferation rate of aortic SMC cultures, as monitored by bromodeoxyuridine incorporation. Taken together, these data suggest that OPG acts as an autocrine/paracrine growth factor for vascular SMC, which might contribute to the progression of atherosclerotic lesions.Key words: osteoprotegerin, mesenchymal stem cells, smooth muscle cells, atherosclerosis.A therosclerosis is a form of chronic low-grade inflammation resulting from interaction between modified lipoproteins, monocyte-derived macrophages and vascular smooth muscle cells (SMC) (Libby, 2002). Although the prevalent view is that intimal vascular SMC found in atherosclerotic plaques derive from cells migrating from the tunica media of the same artery (Libby, 2002), accumulating data indicate that also bone marrow (BM) mesenchymal stem cells (MSC), also known as multipotent stromal cells, have the potential to migrate in sites of vascular injury or inflammation and to differentiate into vascular SMC (Hillebrands et al., 2001, Li et al., 2001).Several studies have clearly demonstrated that the serum levels of the soluble member of the TNF-receptor super-family osteoprotegerin (OPG) are elevated in patients with coronary or carotid artery disease, especially those with clinically unstable atherosclerotic plaques (Jono et al., 2002, Schoppet et al., 2003, Secchiero et al., 2006a, Shin et al., 2006, Abedin et al., 2007, Avignon et al., 2007, Gulbiken et al., 2007, Kadoglu et al., 2008a, Omland et al., 2008). Despite the fact that neither the cellular source nor the physiological and pathological effects of elevated serum levels of OPG are well understood, a possible pathogenic link between elevated levels of OPG and inflammation has been suggested by recent in vitro studies of our and others research groups (Zauli et al., 2007, Mangan et al. 2007).Therefore, in order to assess the potential contribution of MSC in the pathogenesis of atherosclerosis, we have evaluated the release of OPG in the culture supernatants of BM-derived MSC differentiating along the vascular SMC or endothelial cell (EC) lineages. In addition, we have investigated the effect of recombinant human OPG on aortic SMC proliferation.     

Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder caused by a CCTG repeat expansion in intron 1 of ZNF9 gene. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In situ hybridization represents a rapid and sensitive method to obtain a definitive diagnosis in few hours, since it allows the direct visualization of the mutant mRNA foci on skeletal muscle sections. This approach makes the muscle biopsy an important tool for definitive diagnosis of DM2. Consequently, a rapid freezing at ultra cold temperature and a good storage of muscle specimens are essential to avoid morphologic alterations and nucleic acids degradation. However incorrect freezing or thawing may accidentally occur. In this work we report that fluorescence in situ hybridization may be applied on improperly frozen or inappropriately stored muscle biopsies since foci of mutant mRNA are well preserved and can still be detected in muscle sections no more useful for histopathological evaluation.Key words: myotonic dystrophy type 2, defrozen muscle biopsy, fluorescence, in situ hybridization, ribonuclear inclusions.Myotonic dystrophy type 2 (DM2) is a neuromuscular disorder due to the unstable (CCTG)n repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene on chromosome 3q21.3 (Liquori et al. 2001). Mutant ZNF9 pre-mRNA is spliced and polyadenylated, and the mRNA is exported to the cytoplasm where normal levels of ZNF9 protein expression occur (Botta et al., 2006; Margolis et al. 2006); however, the expanded repeats remain in cell nuclei as ribonuclear inclusions (Liquori et al. 2001). The DM2 ribonuclear inclusions contain only the CCUG repeat sequence derived from intron 1 but with no detectable flanking intronic RNA (Margolis et al. 2006). CCUG-containing mutant mRNAs form double-stranded hairpin loop structures that bind specific RNA-binding proteins such as muscle-blind-like proteins (MBNLs) that colocalize with ribonuclear inclusions in myonuclei (Mankodi et al., 2001; Fardaei et al., 2002). Sequestration of these proteins which are regulators of alternative splicing, alters the splicing of several pre-mRNA (reviewed by Osborne and Thornton, 2006) such as the insulin receptor (IR) and the chloride channel (ClC1) (Savkur et al., 2004; Charlet et al., 2002; Mankodi et al., 2002). Alterations in IR splicing leads to insulin insensitivity and predisposition to diabetes (Savkur et al. 2004) and alterations in ClC1 splicing results in electrical myotonia (Charlet et al., 2002; Mankodi et al., 2002). Conventional Southern blot analysis is not adequate for a definitive molecular diagnosis in DM2 due to the extremely large size and somatic instability of the expansion mutation (Liquori et al., 2001; Bachinski et al., 2003). The extraordinary somatic instability complicates the analysis of genotype-phenotype correlations including those in the effect of the gender of transmitting parents and anticipation. The copy number of DM2 CCTG is below 30 in phenotypically normal individuals and up 11.000 in patients (Day and Ranum, 2005). A complex genotyping diagnostic procedure is now commonly used consisting of a three-step molecular protocol (Day et al., 2003; Udd et al., 2003). However, a more practical tool to obtain a definitive diagnosis in few hours is represented by in situ hybridization which detects ribonuclear inclusions in cell nuclei of muscle fibers (Cardani et al., 2004; Sallinen et al., 2004). This approach makes muscle biopsy an essential tool for DM2 diagnosis. For this reason muscle specimens should be sent fresh, for rapid freezing, from the operating room to the pathology laboratory.To avoid RNA degradation, biopsies require special precautions with handling of material, such as immediate freezing of fresh tissues, because retrospective genetic analysis is impaired by conventional tissue processing techniques. However, many small hospitals are ill-equipped for snap freezing which requires access to liquid nitrogen or dry ice; thus, frequently outside hospitals provide specimens that are obscured with freeze artefacts because they either were submitted incorrectly or were improperly frozen, at the point of origin prior to shipment. Moreover, an accidental tissue thawing and refreezing may occur (for example power failure of the freezer) causing severe tissue damages and possible RNA degradation.Here we report our experience on DM2 muscle biopsies improperly preserved: these were no more useful for a histopathological analysis since they showed evident morphologic artefacts, but they proved to be still suitable for diagnosis by fluorescence in situ hybridization (FISH) since ribonuclear inclusions were preserved and still detectable on muscle sections.     

R type anion channel: A multifunctional channel seeking its molecular identity

Plant genomes code for channels involved in the transport of cations, anions and uncharged molecules through membranes. Although the molecular identity of channels for cations and uncharged molecules has progressed rapidly in the recent years, the molecular identity of anion channels has lagged behind. Electrophysiological studies have identified S-type (slow) and R-type (rapid) anion channels. In this brief review, we summarize the proposed functions of the R-type anion channels which, like the S-type, were first characterized by electrophysiology over 20 years ago, but unlike the S-type, have still yet to be cloned. We show that the R-type channel can play multiple roles.Key words: R-type anion channel, nitrate, sulphate, guard cell, action potentialAnion channels play a central role in signal transduction, nutrient transport and cell turgor regulation.¹ By far, their function was particularly well investigated in the guard cells of stomata using a combination of electrophysiological, pharmacological and genetic tools. In this system, anion channel activation was shown to be one of the limiting steps in the loss of cell turgor leading to stomatal closure.² In algal cells, anion channels were shown to contribute to membrane excitability through the generation of action potential.^1,3With the burst of molecular biology in the nineties, the genes coding for plant ion channels started to be unveiled. The first channel gene to be cloned in plant was the shaker-like potassium channel identified in a yeast functional expression screen.^4,5 More than ten years later, TaALMT1 and AtCLCa were characterized as the first members of two important anion channel families.^6,7 This growing group of newly identified channels, accounting for electrophysiological activity described long ago, includes the MSLs anion selective mechanosensitive channels.⁸ Recently, the well known S-type channel has been finally recognized to be encoded by members of the SLAC1 (and other SLAH) family (Slow Anion Channel-Associated 1).⁹ In agreement with electrophysiological data,^10–13 it requires phosphorylation by a Protein Kinase in order to be functional.^14,15 In contrast, the molecular identity of the R-type anion channel remains unknown. Therefore, this candidate, which has been functionally known since twenty years, remains the next challenge for plant channel physiologists.     

Cytokeratin 20-positive hepatocellular carcinoma

The differential diagnosis between hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and metastatic colorectal adenocarcinoma (MCA) may be difficult when only based on morphology. For this purpose immunohistochemical analyses are often required, utilizing antibodies directed against CK8-18, Hep-Par1, glypican 3, CK7, CK19, CK20. Here we report a case of a 65-year-old man who presented with a clinical picture of decompensated cirrhosis. Ultrasonography revealed two nodular areas in the right liver lobe. Liver needle biopsy revealed micro-macronodular cirrhosis associated with HCC with trabecular and pseudoglandular patterns. Immunohistochemically, tumour cells were diffusely positive for CK8-18 and also diffusely immunostained by glypican 3 and Hep-Par1. Interestingly, a diffuse and strong staining for CK20 was detected in the vast majority of tumor cells, particularly in the areas showing a pseudo-glandular pattern. No immunostaining for CK7 and CK19 was found in the tumor cells. The tumor behaved aggressively, with a rapid diffusion to the whole liver. The patient died from the disease few months after presentation. These findings underline that the interpretation of the expression of CK20 alone in the differential diagnosis among HCC, CC and MCA should be done with caution because a diffuse immunoreactivity for CK20 alone may not rule out the diagnosis of HCC.Key words: hepatocellular carcinoma, cholangiocarcinoma, metastatic colorectal carcinomas, CK20.The differential diagnosis between hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and metastatic colorectal adenocarcinoma (MCA) may be difficult when only based on morphology (Terracciano et al., 2003).In fact, a subset of extrahepatic adenocarcinomas of different origin may show a solid “hepatoid” pattern virtually indistinguishable from HCC (Porcell et al., 2000). On the other hand, the undifferentiated form of HCC may mimic poorly differentiated tumors of different origin, while its tubular and adenoid variants may be indistinguishable from CC or from MCA.In these cases, immunohistochemical analyses are often required (Stroescu et al., 2006).The panel of antibodies utilized to solve this differential diagnosis includes: CK8-18 (Porcell et al., 2000) Hep-Par1 (Leong et al., 1998) (Zimmerman et al., 2001), glypican 3 (GPC3) (Yamauchi et al., 2005) (Capurro et al., 2003), CK7 (Maeda et al., 1996) (Chu et al., 2000), CK20 (Faa G et al., 1998), CK19, CEA and Alpha-fetoprotein (Onofre et al., 2007) (Lau et al., 2002).Immunoreactivity of tumour cells for CK8-18, Hep-Par 1 and GPC3 is considered suggestive of HCC; a diffuse immunoreactivity for CK7 and CK19 is in favour of the diagnosis of CC; a diffuse positivity for CK20 and negativity for CK7 are normally associated with MCA.Here we report a case of HCC with a peculiar immunohistochemical profile, characterized by the association of the typical immunoreactivity of HCC with a diffuse and strong positivity for CK20, generally considered typical of MCA.     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.