The calcium-activated chloride channel-associated protein rCLCA2 is expressed throughout rat epidermis, facilitates apoptosis and is downmodulated by UVB.

The rodent chloride channel regulatory proteins mCLCA2 and its porcine and human homologues pCLCA2 and hCLCA2 are expressed in keratinocytes but their localization and significance in the epidermis have remained elusive. hCLCA2 regulates cancer cell migration, invasion and apoptosis, and its loss predicts poor prognosis in many tumors. Here, we studied the influences of epidermal maturation and UV-irradiation (UVR) on rCLCA2 (previous rCLCA5) expression in cultured rat epidermal keratinocytes (REK) and correlated the results with mCLCA2 expression in mouse skin in vivo. Furthermore, we explored the influence of rCLCA2 silencing on UVR-induced apoptosis. rClca2 mRNA was strongly expressed in REK cells, and its level in organotypic cultures remained unchanged during the epidermal maturation process from a single cell layer to fully differentiated, stratified cultures. Immunostaining confirmed its uniform localization throughout the epidermal layers in REK cultures and in rat skin. A single dose of UVR modestly downregulated rClca2 expression in organotypic REK cultures. The immunohistochemical staining showed that CLCA2 localized in basal and spinous layers also in mouse skin, and repeated UVR induced its partial loss. Interestingly, silencing of rCLCA2 reduced the number of apoptotic cells induced by UVR, suggesting that by facilitating apoptosis, CLCA2 may protect keratinocytes against the risk of malignancy posed by UVB-induced corrupt DNA.

Effects of mutations in the post-translational modification sites on the trafficking of hyaluronan synthase 2 (HAS2).

Vesicular trafficking of hyaluronan synthases (HAS1-3) from endoplasmic reticulum (ER) through Golgi to plasma membrane (PM), and either back to endosomes and lysosomes, or out into extracellular vesicles, is important for their activities. We studied how post-translational modifications affect the trafficking of HAS2 by mutagenesis of the sites of ubiquitination (K190R), phosphorylation (T110A) and O-GlcNAcylation (S221A), using Dendra2- and EGFP-HAS2 transfected into COS1 cells. Confocal microscopy showed HAS2 wild type (wt) and its K190R and S221A mutants in ER, Golgi and extracellular vesicles, while the T110A mutant remained mostly in the ER. HA synthesis was reduced by S221A, while completely blocked by K190R and T110A. Cell-surface biotinylation indicated that T110A was absent from PM, while S221A was close to the level of wt, and K190R was increased in PM. TIRF microscopy analysis gave similar results. Rab10 silencing increased HA secretion by HAS2, likely by inhibiting endocytosis of the enzyme from PM, as reported before for HAS3. Green-to-red photo-conversion of Dendra2-HAS2 constructs suggested slower decay of K190R and S221A than HAS2 wt, while T110A was barely degraded at all. S221D and S221E, the phosphomimetic mutants of this site, decayed faster and blocked hyaluronan synthesis, suggesting alternative O-GlcNAc/-PO4 substitution to regulate the stability of the enzyme. Probing the role of dynamic O-GlcNAcylation at S221 by adding glucosamine increased the half-life of only HAS2 wt. The Dendra2·HAS2 disappearance from Golgi was slower for K190R. Of the two inactive constructs, K190R co-transfected with HAS2 wt suppressed, whereas T110A had no effect on HA synthesis. Interestingly, the HAS2-stimulated shedding of extracellular vesicles was dependent on HAS residence in PM but independent of HA synthesis. The results indicate that post-translational modifications control the trafficking of HAS2, and that trafficking is an integral part of the post-translational regulation of HAS2 activity.

Hyaluronan metabolism enhanced during epidermal differentiation is suppressed by vitamin C.

BACKGROUND: Hyaluronan is a large, linear glycosaminoglycan present throughout the narrow extracellular space of the vital epidermis. Increased hyaluronan metabolism takes place in epidermal hypertrophy, wound healing and cancer. Hyaluronan is produced by hyaluronan synthases and catabolized by hyaluronidases, reactive oxygen species and KIAA1199. OBJECTIVES: To investigate the changes in hyaluronan metabolism during epidermal stratification and maturation, and the impact of vitamin C on these events. METHODS: Hyaluronan synthesis and expression of the hyaluronan-related genes were analysed during epidermal maturation from a simple epithelium to a fully differentiated epidermis in organotypic cultures of rat epidermal keratinocytes using quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blotting, in the presence and absence of vitamin C. RESULTS: With epidermal stratification, both the production and the degradation of hyaluronan were enhanced, resulting in an increase of hyaluronan fragments of various sizes. While the mRNA levels of Has3 and KIAA1199 remained stable during the maturation, Has1, Has2 and Hyal2 showed a transient upregulation during stratification, Hyal1 transcription remained permanently increased and transcription of the hyaluronan receptor, Cd44, decreased. At maturation, vitamin C downregulated Has2, Hyal2 and Cd44, whereas it increased high-molecular-mass hyaluronan in the epidermis, and reduced small fragments in the medium, suggesting stabilization of epidermal hyaluronan. CONCLUSIONS: Epidermal stratification and maturation is associated with enhanced hyaluronan turnover, and release of large amounts of hyaluronan fragments. The high turnover is suppressed by vitamin C, which is suggested to enhance normal epidermal differentiation in part through its effect on hyaluronan.

Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice.

Wnt signaling is a conserved pathway involved in expansion of neural progenitors and lineage specification during development. However, the role of Wnt signaling in the post-stroke brain has not been well-elucidated. We hypothesized that Wnt-3a would play an important role for neurogenesis and brain repair. Adult male mice were subjected to a focal ischemic stroke targeting the sensorimotor cortex. Mice that received Wnt-3a (2 µg/kg/day, 1 h after stroke and once a day for the next 2 days, intranasal delivery) had reduced infarct volume compared to stroke controls. Wnt-3a intranasal treatment of seven days upregulated the expression of brain-derived growth factor (BDNF), increased the proliferation and migration of neuroblasts from the subventricular zone (SVZ), resulting in increased numbers of newly formed neurons and endothelial cells in the peri-infarct zone. Both the molecular and cellular effects of Wnt-3a were blocked by the Wnt specific inhibitors XAV-939 or Dkk-1. In functional assays, Wnt-3a treatment enhanced the local cerebral blood flow (LCBF) in the peri-infarct, as well as improved sensorimotor functions in a battery of behavioral tests. Together, our data demonstrates that the Wnt-3a signaling can act as a dual neuroprotective and regenerative factor for the treatment of ischemic stroke.

Regulatory role of the JNK-STAT1/3 signaling in neuronal differentiation of cultured mouse embryonic stem cells.

Stem cell transplantation therapy has provided promising hope for the treatment of a variety of neurodegenerative disorders. Among challenges in developing disease-specific stem cell therapies, identification of key regulatory signals for neuronal differentiation is an essential and critical issue that remains to be resolved. Several lines of evidence suggest that JNK, also known as SAPK, is involved in neuronal differentiation and neural plasticity. It may also play a role in neurite outgrowth during neuronal development. In cultured mouse embryonic stem (ES) cells, we test the hypothesis that the JNK pathway is required for neuronal differentiation. After neural induction, the cells were plated and underwent differentiation for up to 5 days. Western blot analysis showed a dramatic increase in phosphorylated JNKs at 1-5 days after plating. The phosphorylation of JNK subsequently induced activation of STAT1 and STAT3 that lead to expressions of GAP-43, neurofilament, ßIII-tubulin, and synaptophysin. NeuN-colabelled with DCX, a marker for neuroblast, was enhanced by JNK signaling. Neuronal differentiation of ES cells was attenuated by treatment with SP600125, which inhibited the JNK activation and decreased the activation of STAT1 and STAT3, and consequently suppressed the expressions of GAP-43, neurofilament, ßIII-tubulin, and the secretion of VEGF. Data from immunocytochemistry indicated that the nuclear translocation of STAT3 was reduced, and neurites of ES-derived neurons were shorter after treatment with SP600125 compared with control cells. These results suggest that the JNK-STAT3 pathway is a key regulator required for early neuronal differentiation of mouse ES cells. Further investigation on expression of JNK isoforms showed that JNK-3 was significantly upregulated during the differentiation stage, while JNK-1 and JNK-2 levels decreased. Our study provided interesting information on JNK functions during ES cell neuronal differentiation.

rClca2 is associated with epidermal differentiation and is strongly downregulated by ultraviolet radiation.

BACKGROUND: Excessive skin exposure to solar radiation damages proteins and DNA, ultimately leading to skin ageing and cancers. OBJECTIVES: To identify new ultraviolet B (UVB) target genes to understand the mechanisms behind the detrimental effects of UVB. METHODS: Organotypic, stratified cultures of rat keratinocytes were exposed to UVB and analysed using a genome-wide expression array, quantitative real-time polymerase chain reaction and histology. The most downregulated gene, rClca2, was further characterized in rat keratinocytes and mouse skin models. RESULTS: A single, 30 mJ cm(-2) dose of broadband UVB proved effective in the organotypic epidermal culture. The expression of 627 genes was changed 24 h postirradiation. In silico analysis of the data indicated activation of DNA repair, metabolism, cell cycle control and amino acid metabolism, but only limited inflammation under these conditions. We selected for further investigation the most downregulated gene, rClca2, previously suggested to regulate keratinocyte differentiation and adhesion, and found that UVB caused a long-lasting downregulation in its expression. Both the rClca2 full-length isoform (expressed in the differentiating cells) and the truncated isoform (expressed in the basal layers) were reduced by UVB. Immunohistochemistry of mouse skin samples with isoform-specific antibodies showed a similar, epidermal differentiation-related pattern. In mouse specimens exposed to chronic ultraviolet radiation (UVR) the staining intensities were reduced and the differentiation-related isoform was disturbed in the hyperplastic and carcinomatous areas induced by UVR. CONCLUSIONS: The data show that rClca2 is a novel UVB target gene and suggest that it might play a role in epidermal differentiation and UV-dependent skin malignancies.

Hyaluronan synthase 1 (HAS1) produces a cytokine-and glucose-inducible, CD44-dependent cell surface coat.

Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER-Golgi-plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1ß, TNF-α, or TGF-ß. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.

Delayed intranasal delivery of hypoxic-preconditioned bone marrow mesenchymal stem cells enhanced cell homing and therapeutic benefits after ischemic stroke in mice.

Stem cell transplantation therapy has emerged as a potential treatment for ischemic stroke and other neurodegenerative diseases. Effective delivery of exogenous cells and homing of these cells to the lesion region, however, have been challenging issues that hinder the efficacy and efficiency of cell-based therapy. In the present investigation, we tested a delayed treatment of noninvasive and brain-targeted intranasal delivery of bone marrow mesenchymal stem cells (BMSCs) in a mouse focal cerebral ischemia model. The investigation tested the feasibility and effectiveness of intranasal delivery of BMSCs to the ischemic cortex. Hypoxia preconditioning (HP) of BMSCs was performed before transplantation in order to promote their survival, migration, and homing to the ischemic brain region after intranasal transplantation. Hoechst dye-labeled normoxic- or hypoxic-pretreated BMSCs (1 × 10(6) cells/animal) were delivered intranasally 24 h after stroke. Cells reached the ischemic cortex and deposited outside of vasculatures as early as 1.5 h after administration. HP-treated BMSCs (HP-BMSCs) showed a higher level of expression of proteins associated with migration, including CXC chemokine receptor type 4 (CXCR4), matrix metalloproteinase 2 (MMP-2), and MMP-9. HP-BMSCs exhibited enhanced migratory capacities in vitro and dramatically enhanced homing efficiency to the infarct cortex when compared with normoxic cultured BMSCs (N-BMSCs). Three days after transplantation and 4 days after stroke, both N-BMSCs and HP-BMSCs decreased cell death in the peri-infarct region; significant neuroprotection of reduced infarct volume was seen in mice that received HP-BMSCs. In adhesive removal test of sensorimotor functional assay performed 3 days after transplantation, HP-BMSC-treated mice performed significantly better than N-BMSC- and vehicle-treated animals. These data suggest that delayed intranasal administration of stem cells is feasible in the treatment of stroke and hypoxic preconditioning of transplanted cells, significantly enhances cell's homing to the ischemic region, and optimizes the therapeutic efficacy.

Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation.

Transplantation using stem cells including bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the heart and brain. The migration capability of transplanted cells is a critical cellular function for tissue repair. Based on our recent observations that hypoxic preconditioning (HP) has multiple benefits in improving stem cell therapy and that the potassium Kv2.1 channel acts as a promoter for focal adhesion kinase (FAK) activation and cell motility, the present investigation tested the hypothesis that HP treatment can increase BMSC migration via the mechanism of increased Kv2.1 expression and FAK activities. BMSCs derived from green fluorescent protein-transgenic mice were treated under either normoxic (N-BMSC) or hypoxic (0.5% O(2)) (HP-BMSC) conditions for 24 h. Western blot analysis showed HP selectively upregulated Kv2.1 expression while leaving other K(+) channels, such as Kv1.5 and Kv1.4, unaffected. Compared with normoxic controls, significantly larger outward delayed rectifier K(+) currents were recorded in HP-BMSCs. HP enhanced BMSC migration/homing activities in vitro and after intravenous transplantation into rats subjected to permanent myocardial infarction (MI). The HP-promoted BMSC migration was inhibited by either blocking K(+) channels or knocking down Kv2.1. Supporting a relationship among HP, Kv2.1, and FAK activation, HP increased phosphorylation of FAK(397) and FAK(576/577), and this effect was antagonized by blocking K(+) channels. These findings provide novel evidence that HP enhances the ability of BMSCs to migrate and home to the injured region; this effect is mediated through a regulatory role of Kv2.1 on FAK phosphorylation/activation.

Hyaluronan in human malignancies.

Hyaluronan, a major macropolysaccharide in the extracellular matrix of connective tissues, is intimately involved in the biology of cancer. Hyaluronan accumulates into the stroma of various human tumors and modulates intracellular signaling pathways, cell proliferation, motility and invasive properties of malignant cells. Experimental and clinicopathological evidence highlights the importance of hyaluronan in tumor growth and metastasis. A high stromal hyaluronan content is associated with poorly differentiated tumors and aggressive clinical behavior in human adenocarcinomas. Instead, the squamous cell carcinomas and malignant melanomas tend to have a reduced hyaluronan content. In addition to the stroma-cancer cell interaction, hyaluronan can influence stromal cell recruitment, tumor angiogenesis and epithelial-mesenchymal transition. Hyaluronan receptors, hyaluronan synthases and hyaluronan degrading enzymes, hyaluronidases, are involved in the modulation of cancer progression, depending on the tumor type. Furthermore, intracellular signaling and angiogenesis are affected by the degradation products of hyaluronan. Hyaluronan has also therapeutic implications since it is involved in multidrug resistance.

Depletion of cell surface CD44 in nonmelanoma skin tumours is associated with increased expression of matrix metalloproteinase 7.

BACKGROUND: Expression of matrix metalloproteinase (MMP)-7 and MMP-9 is low in the normal epidermis and is induced by physiological processes such as wound healing, but also malignant transformation of epidermal cells. The activity of both MMPs has been associated with the hyaluronan (HA) receptor CD44. We previously reported that the levels of CD44 and HA differ between the two types of epidermal tumours, basal (BCC) and squamous cell carcinoma (SCC), as well as between different grades of SCC. OBJECTIVES: To investigate if the immunostaining patterns of MMP-7 and MMP-9 correlate to those of CD44 and HA in BCC and SCC. METHODS: Paraffin sections from 71 BCCs, 21 in situ SCCs and 27 SCCs were immunostained for MMP-7 and -9. RESULTS: Positive immunostaining for MMP-7 and MMP-9 was found in tumour cells of both BCC and SCC, while the staining intensity tended to be stronger in SCC. The staining intensity of MMP-7 was inversely correlated with that of CD44 in both tumour types. In well-differentiated SCC, the intensity of MMP-7 was generally weak, while CD44 staining was strong and homogeneously distributed. In poorly differentiated SCC, an increase in MMP-7 was seen, and the staining intensity of CD44 became weak and was locally absent. No correlation was seen between MMP-9 and CD44 or either of the two MMPs and HA. CONCLUSIONS: Our results show that in nonmelanoma skin tumours MMP-7 and -9 are present in the tumour cells, and suggest a link between MMP-7 activity and the depletion of cell surface CD44.

Trends in the exploration of anticancer targets and strategies in enhancing the efficacy of drug targeting.

A number of therapeutic targets have been explored for developing anticancer drugs. Continuous efforts have been directed at the discovery of new targets as well as the improvement of therapeutic efficacy of agents directed at explored targets. There are 84 and 488 targets of marketed and investigational drugs for the treatment of cancer or cancer related illness. Analysis of these targets, particularly those of drugs in clinical trials and US patents, provides useful information and perspectives about the trends, strategies and progresses in targeting key cancer-related processes and in overcoming the difficulties in developing efficacious drugs against these targets. The efficacy of anticancer drugs directed at these targets is frequently compromised by counteractive molecular interactions and network crosstalk, negative and adverse secondary effects of drugs, and undesired ADMET profiles. Multi-component therapies directed at multiple targets and improved drug targeting methods are being explored for alleviating these efficacy-reducing processes. Investigation of the modes of actions of these combinations and targeting methods offers clues to aid the development of more effective anticancer therapies.

Prognostic significance of extracellular matrix metalloproteinase inducer and matrix metalloproteinase 2 in epithelial ovarian cancer.

AIMS: We investigated the prognostic significance of extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase 2 (MMP-2) in epithelial ovarian cancer as well as their relation to hyaluronan (HA) expression. METHODS: The expression of EMMPRIN and MMP-2 was analyzed immunohistochemically in 295 primary epithelial ovarian cancer patients and 67 metastases. RESULTS: A low membranous EMMPRIN expression was detected more often in serous tumors than in other types (p < 0.0005) and it was associated with tumors of advanced stage (p = 0.012) or with a large primary residual (p = 0.011). A low expression of MMP-2 in cancer cells was associated with a high histologic grade (grade 3) of the tumor (p = 0.005) and endometrioid type of tumors (p < 0.0005). Stromal MMP-2 expression was significantly associated with strong stromal HA expression (p = 0.002, r = 0.187). In univariate analysis, 10-year disease-related (DRS) and recurrence-free survivals were significantly better when MMP-2 expression in cancer cells was high (p = 0.0057 and p = 0.0467, respectively). DRS was also better when membranous EMMPRIN expression was high (p = 0.013). In multivariate analysis, strong MMP-2 in cancer cells (RR = 1.48, CI = 1.07-2.04, p = 0.017) indicated favorable DRS. CONCLUSION: Our results show that EMMPRIN and MMP-2 in cancer cells are significant indicators of a favorable prognosis of epithelial ovarian cancer.

Prognostic significance of matrix metalloproteinase-9 (MMP-9) in epithelial ovarian cancer.

OBJECTIVE: We investigated the expression of matrix metalloproteinase-9 (MMP-9) and its relation to clinicopathologic factors and survival and also to previously analyzed expressions of CD44 and hyaluronan in epithelial ovarian cancer. METHODS: The expression of MMP-9 was analyzed immunohistochemically in 292 primary tumors and their 31 metastases. RESULTS: A low proportion of strong MMP-9 expression in cancer cells and high stromal MMP-9 expression correlated with advanced stage of the tumor (p=0.003, p=0.02, respectively). Stromal MMP-9 expression significantly correlated with hyaluronan positivity (p<0.0005), whereas MMP-9 did not correlate with CD44. In univariate analysis, a longer 10-year disease-related survival (DRS) was found in patients with a high proportion of MMP-9 or strong MMP-9 expression in cancer cells (p=0.02, p=0.05, respectively). However, high stromal expression of MMP-9 indicated short DRS (p=0.01). In multivariate analysis of all patients, MMP-9 expressing cancer or stromal cells were not independent prognostic factors, while in FIGO stage I patients a high percentage of MMP-9 positive cancer cells was associated with long DRS (p=0.008). CONCLUSION: These data suggest that MMP-9 has a dual role in tumor progression, acting against tumor advancement when in tumor epithelium and promoting tumor progression while in the stroma.

Quantitative determination of hyaluronan content in cerebral aneurysms by digital densitometry.

OBJECT: Hyaluronan (HA) is a highly hydrated macromolecule; it is one of the essential components of the extracellular matrix (ECM) of the arteries and plays an important role in maintaining the biomechanical features of blood vessels. Although the potential contribution of HA in aneurysms of different vessels has been studied intensively, no data are available about the alteration of the HA content in the extracellular matrix of intracranial aneurysms. The aim of the study was to determine the hyaluronan content in the wall of human cerebral arteries. METHODS: A biotinylated aggrecan fragment that binds specifically to HA was used to stain samples from cerebral aneurysms (n = 11) to compare the HA content to non-aneurysmal arteries of patients who had intracranial aneurysm (n = 11), and to histologically normal arteries of patients who had expired from non-vascular diseases (n = 14). Digital microscopic densitometry was used for the quantitative analysis of the hyaluronan content in these samples. RESULTS: The highest level (169.5 +/- 7.9) was detected in aneurysms, while the HA-level of non-aneurysmal vessels was lower (130.2 +/- 16.8). Both vessel groups contained significantly higher HA than the normal cerebral arteries (32.9 +/- 2.1). CONCLUSIONS: Results suggest that an elevated hyaluronan level in the extracellular matrix may affect the cerebral arterial wall architecture. It is reasonable to suppose that the increased hyaluronan content creates a viscoelastic ECM which might improve the biomechanical resistance of the thinned vessel wall.

Expression of CD 44 s, CD 44 v 3 and CD 44 v 6 in benign and malignant breast lesions: correlation and colocalization with hyaluronan.

AIMS: To examine the expression of CD 44 s, CD 44 v 3 and CD 44 v 6 in breast lesions, and to correlate it with the expression of hyaluronan (HA). METHODS AND RESULTS: CD 44 expression was studied in 75 breast tissue samples, consisting of benign, premalignant and malignant breast lesions, using immunohistochemistry. CD 44 s, but not CD 44 v 3 or CD 44 v 6, was found in the stromal cells, and it was similar in benign and malignant tumours. In benign lesions CD 4 v 6 was detected in 20-30% of the ductal epithelial cells, while C 44 v 3 and CD 44 s were not expressed. CD 44 s, CD 44 v 3 and CD 44 v 6 were all up-regulated in the in situ carcinomas and invasive carcinomas. The level of CD 44 expression in carcinoma cells did not correlate with the type or differentiation of the tumours. CD 44 and HA expression levels were not closely linked in the benign or malignant breast lesions, because HA was overexpressed later in breast cancer progression than CD 44. However, in breast carcinomas CD 44 and HA positivity was often found in the same areas of the sections, and the dual staining confirmed actual colocalization of CD 44 s and HA in the same cells. CONCLUSIONS: CD 44 s, CD 44 v 3 and CD 44 v 6 are up-regulated earlier than HA in breast carcinoma progression, and in later stages they often colocalize with cell surface HA.

Elevated expression of hyaluronan and its CD44 receptor in the duodenal mucosa of coeliac patients.

AIMS: Since hyaluronan (HA) metabolism is disturbed in some malignant tumours and in inflammatory diseases, we analysed HA and its receptor CD44 as well as the expression of the Ki67 nuclear protein, a marker of cell proliferation, in histological sections of duodenal biopsies of coeliac disease patients and controls. METHODS AND RESULTS: The study group consisted of 52 patients with coeliac disease in remission, 40 patients with newly diagnosed disease and 10 healthy control subjects. HA was detected with a specific biotinylated probe prepared from cartilage aggrecan and link protein, and CD44 with an antibody recognizing all forms of CD44 and another specific for its v6 variant. For the expression of the nuclear protein, monoclonal antibody MIB-1 was used. The percentage of HA-positive cells in surface epithelium was higher in newly diagnosed patients (13%) compared with patients in remission (11%) and controls (2%). In addition, HA intensity in the lamina propria was decreased in the newly diagnosed patients. In patients with active disease, 22-26% of the surface epithelium was CD44+, whereas the corresponding figure in patients in remission was 5%, and that of controls 1%. The more intensive MIB-1 labelling in the duodenal epithelium of coeliac patients without treatment was normalized after gluten-free diet. CONCLUSIONS: The HA-positive coat on surface epithelium seen even in patients in remission suggests persistent or even permanent changes in the epithelial permeability barrier in coeliac disease.

Reduced expression of hyaluronan is a strong indicator of poor survival in oral squamous cell carcinoma.

Several malignant tumours accumulate hyaluronan (HA), a matrix component suggested to promote cancer cell growth and migration. The expression and prognostic value of HA was analysed in a cohort of 151 oral squamous cell carcinoma (SCC) patients with adequate archival tumour material and follow-up data. The tumour samples were stained using a biotinylated HA-specific probe. Normal squamous epithelium showed a strong and homogeneously distributed staining for HA. The most superficial layers were HA-negative. In moderate (n=11) and high grade (n=16) dysplasias an irregular HA staining was observed around invasive cancer. Malignant transformation in oral squamous cell epithelium changed the staining toward irregular with focal reduction of HA. The well (n=92) or moderately differentiated (n=47) carcinomas had a strong HA staining intensity. In poorly differentiated tumours (n=12) the HA staining was weaker and mainly intracellular. The stromal tissue showed usually moderate (n=69) or strong (n=67) HA staining intensity with no statistically significant correlation with the degree of tumour differentiation. At the end of the follow-up (median 52 months) 66 (43%) patients had died because of an oral SCC. A significant difference in overall survival (OS) and disease free survival (DFS) (P=0.0002 and 0.0020, respectively) was noticed between the patients with the different epithelial staining patterns for HA. The reduction of HA staining was associated with poor survival. In Cox's multivariate analysis HA staining was a significant independent predictor of OS (P=0.011) and DFS (P=0.013). These results suggest that HA is a prognostic marker in oral squamous cell carcinoma.

Hyaluronan, CD44 and versican in epidermal keratinocyte tumours.

BACKGROUND: The high molecular weight polysaccharide hyaluronan is a major component of the extracellular matrix between the vital cells of human skin epidermis. The levels of hyaluronan, and those of the hyaluronan receptor CD44 and the hyaluronan binding proteoglycan versican, correlate with the aggressiveness of different human carcinomas of epithelial origin. OBJECTIVES: To study skin keratinocyte tumours for the expression of hyaluronan, the hyaluronan receptor CD44 and the hyaluronan binding proteoglycan versican. METHODS: Paraffin-embedded sections of 114 basal cell carcinomas (BCC), 31 in situ carcinomas (ISC) and 35 squamous cell carcinomas (SCC) were stained with a hyaluronan specific probe, biotinylated hyaluronan binding complex, and with monoclonal antibodies against CD44 and versican. RESULTS: Compared with normal epidermis, ISC and well differentiated SCCs showed an enhanced hyaluronan signal on carcinoma cells while CD44 expression level resembled that of normal skin. Less differentiated SCCs showed reduced and irregular expression of both hyaluronan and CD44 on carcinoma cells. In BCCs, hyaluronan and CD44 signals were absent or very low on the surface of carcinoma cells. However, hyaluronan was frequently present on BCC cell nuclei, a feature completely absent in ISC, SCC and normal epidermis. An accumulation of hyaluronan in the connective tissue stroma around the tumour was more frequent in SCCs than BCCs. Versican staining was positive around hair follicles and dermal blood vessels of normal skin. Peritumoral versican signal was present in a part of the BCCs but not in other tumours. CONCLUSIONS: The completely different hyaluronan and CD44 expression patterns in BCC and SCC probably reflect the different origins of the tumours, with BCC an undifferentiated keratinocyte and SCC a keratinocyte at an early stage in the differentiation pathway. The difference in hyaluronan and CD44 expression between these tumours may also contribute to the difference in their capacity to metastasize.

Strong Stromal Hyaluronan Expression Is Associated with PSA Recurrence in Local Prostate Cancer.

OBJECTIVE: HA (hyaluronan) is involved in cell migration, differentiation and cell proliferation, which all are essential to tumour growth. In addition, the cell surface receptor of HA, CD44, is important in cancer cell adhesion, cell migration and tumour neovascularisation. We studied the expression of HA and CD44 and their relationship with other prognostic factors and prostate-specific antigen (PSA) recurrence in local prostate cancer (PC). MATERIALS AND METHODS: 77 PC patients treated with radical prostatectomy were followed-up for a mean of 4 years. HA was detected by using a HA specific probe and CD44 expression was analysed by conventional immunohistochemistry. RESULTS: All specimens expressed HA in tumour stroma and 78% (60/77) of the tumours showed strong stromal expression of HA. The fraction of positively stained specimens for CD44 was 66% (51/77). The strong stromal HA expression was related to perineural infiltration (p = 0.001) and capsule invasion (p = 0.05). No correlation was demonstrated between the stromal HA expression and CD44 expression, preoperative PSA, clinical or pathological T classification, pN status, Gleason grade, seminal vesicle invasion or surgical margin invasion. Reduced CD44 expression was related only to preoperative PSA level (p = 0.008). The PSA recurrence was predicted by strong stromal HA expression, pT classification, seminal vesicle invasion, capsule invasion and surgical margin invasion (p