Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.

BACKGROUND AND AIMS: Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. METHODS: We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. RESULTS: We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. CONCLUSIONS: This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum.

INTRODUCTION: Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally. METHODS: We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified. RESULTS: Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP. CONCLUSIONS: Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.

Recombinant human collagen III gel for transplantation of autologous skin cells in porcine full-thickness wounds.

Complex skin wounds, such as chronic ulcers and deep burns, require lengthy treatments and cause extensive burdens on healthcare and the economy. Use of biomaterials and cell transplantation may improve traditional treatments and promote the healing of difficult-to-treat wounds. In this study, we investigated the use of recombinant human collagen III (rhCol-III) gel as a delivery vehicle for cultured autologous skin cells (keratinocytes only or keratinocyte-fibroblast mixtures). We examined its effect on the healing of full-thickness wounds in a porcine wound-healing model. Two Landrace pigs were used for the study. Fourteen deep dermal wounds were created on the back of each pig with an 8 mm biopsy punch. Syringes containing acellular rhCol-III gel (n = 8) or rhCol-III gel with autologous keratinocytes (n = 8) or rhCol-III gel with autologous keratinocytes and fibroblasts (n = 8) were applied into wounds. Untreated wounds were used as controls for the treatment groups (n = 4). We used rhCol-III gel to manufacture a cell-delivery syringe containing autologous skin cells. In a full-thickness wound-healing model, we observed that rhCol-III gel enhances early granulation tissue formation. Interestingly, we found cell type-dependent differences in the stability of rhCol-III in vivo. Fibroblast-containing gel was effectively removed from the wound, whereas gels without cells or with keratinocytes only remained intact. Our results demonstrate that the properties of rhCol-III gel for skin cell transplantation can be significantly altered in a cell type-dependent manner.

Label-free imaging of adipogenesis by coherent anti-stokes Raman scattering microscopy.

Label-free imaging technologies to monitor the events associated with early, intermediate and late adipogenic differentiation in multipotent mesenchymal stromal cells (MSCs) offer an attractive and convenient alternative to conventional fixative based lipid dyes such as Oil Red O and Sudan Red, fluorescent labels such as LipidTOX, and more indirect methods such as qRT-PCR analyses of specific adipocyte differentiation markers such as peroxisome PPARγ and LPL. Coherent anti-Stokes Raman scattering (CARS) microscopy of live cells is a sensitive and fast imaging method enabling evaluation of the adipogenic differentiation with chemical specificity. CARS microscopy is based on imaging structures of interest by displaying the characteristic intrinsic vibrational contrast of chemical bonds. The method is nontoxic, non-destructive, and minimally invasive, thus presenting a promising method for longitudinal analyses of live cells and tissues. CARS provides a coherently emitted signal that is much stronger than the spontaneous Raman scattering. The anti-Stokes signal is blue shifted from the incident wavelength, thus reducing the non-vibrational background present in most biological materials. In this chapter, we aim to provide a detailed approach on how to induce adipogenic differentiation in MSC cultures, and present our methods related to label-free CARS imaging of the events associated with the adipogenesis.

Toll-like receptors in human chondrocytes and osteoarthritic cartilage.

BACKGROUND AND PURPOSE: Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes. METHODS: We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied. RESULTS: In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166(×) progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9). INTERPRETATION: Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.

Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation.

During adipogenic differentiation human mesenchymal stem cells (hMSC) produce collagen type IV. In immunofluorescence staining differentiating hMSCs started to express collagen type IV when Oil Red O-positive fat droplets appeared intracellularly. Quantitative real time-polymerase chain reaction confirmed progressive increase of collagen type IV α1 and α2 mRNA levels over time, 18.6- and 12.2-fold by day 28, respectively, whereas the copy numbers of α3-α6 mRNAs remained rather stable and low. Type IV collagen was in confocal laser scanning microscopy seen around adipocytes, where also laminins and nidogen were found, suggesting pericellular deposition of all key components of the fully developed basement membrane. Immunofluorescence staining of matrix metalloproteinase-2 (MMP-2, 72 kD type IV collagenase, gelatinase A) and MMP-9 (92 kD type IV collagenase, gelatinase B) disclosed only faint staining of MSCs, but MMP-9 was strongly induced during adipogenesis, whereas MSC supernatants disclosed in zymography pro-MMP-2 and faint pro-MMP-9 bands, which increased over time, with partial conversion of pro-MMP-2 to its active 62 kD form. Differentiation was associated with increasing membrane type 1-MMP/MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) staining, which may enable participation of type IV collagenases in basement membrane remodelling via ternary MT1-MMP/TIMP-2/MMP-2 or -9 complexes, focalizing the fully active enzyme to the cell surface. MMP-9, which increased more in immunofluorescence staining, was perhaps preferentially bound to cell surface and/or remodelling adipocyte basement membrane. These results suggest that upon MSC-adipocyte differentiation collagen type IV synthesis and remodelling become necessary when intracellular accumulation of fat necessitates a dynamically supporting and instructive, partly denatured adipogenic pericellular type IV collagen scaffold.

Improved skin wound epithelialization by topical delivery of soluble factors from fibroblast aggregates.

INTRODUCTION: Timely coverage of an excised burn wound with a split-thickness skin graft, and efficient epithelialization at the donor site wound are key components in the treatment of burn patients. Prompt healing is dependent on paracrine support from underlying dermal connective tissue fibroblasts. STUDY AIM: Using the skin graft donor site in pig as a model for epithelialization, our aim was to evaluate if dermal signals, derived from cultured dermal fibroblast aggregates (Finectra), can promote epidermal regeneration. MATERIALS AND METHODS: Partial-thickness skin wounds were made with a dermatome on the backs of three domestic pigs. After randomization, topical treatment was initiated by application of Finectra (n=6) or factors from standard fibroblast monolayer cultures (n=6) trapped in a slow-clotting fibrin matrix. Saline was applied to contralateral wounds to serve as corresponding untreated controls (n=12). After 3 days, full-thickness skin samples representing the whole wound area were obtained. Histological sections of these samples were analyzed for epithelialization, cell migration from lateral wound edges and hair follicles, as well as for formation of granulation tissue. RESULTS: In response to topical delivery of Finectra, a significant acceleration of epithelialization (p<0.001) across the wound surface as well as from the wound edges was evident. Marked increase in thickness of granulation tissue (p<0.001) was noted in wounds treated with Finectra. Epihelialization originated from adnexal structures in which epithelial islets showed positive staining for cytokeratin-14 and PCNA. CONCLUSION: These data show that the fibroblast aggregate-derived paracrine mediators, Finectra, stimulate epidermal regeneration in vivo.

Inhibiting TRPA1 ion channel reduces loss of cutaneous nerve fiber function in diabetic animals: sustained activation of the TRPA1 channel contributes to the pathogenesis of peripheral diabetic neuropathy.

Peripheral diabetic neuropathy (PDN) is a devastating complication of diabetes mellitus (DM). Here we test the hypothesis that the transient receptor potential ankyrin 1 (TRPA1) ion channel on primary afferent nerve fibers is involved in the pathogenesis of PDN, due to sustained activation by reactive compounds generated in DM. DM was induced by streptozotocin in rats that were treated daily for 28 days with a TRPA1 channel antagonist (Chembridge-5861528) or vehicle. Laser Doppler flow method was used for assessing axon reflex induced by intraplantar injection of a TRPA1 channel agonist (cinnamaldehyde) and immunohistochemistry to assess substance P-like innervation of the skin. In vitro calcium imaging and patch clamp were used to assess whether endogenous TRPA1 agonists (4-hydroxynonenal and methylglyoxal) generated in DM induce sustained activation of the TRPA1 channel. Axon reflex induced by a TRPA1 channel agonist in the plantar skin was suppressed and the number of substance P-like immunoreactive nerve fibers was decreased 4 weeks after induction of DM. Prolonged treatment with Chembridge-5861528 reduced the DM-induced attenuation of the cutaneous axon reflex and loss of substance P-like immunoreactive nerve fibers. Moreover, in vitro calcium imaging and patch clamp results indicated that reactive compounds generated in DM (4-hydroxynonenal and methylglyoxal) produced sustained activations of the TRPA1 channel, a prerequisite for adverse long-term effects. The results indicate that the TRPA1 channel exerts an important role in the pathogenesis of PDN. Blocking the TRPA1 channel provides a selective disease-modifying treatment of PDN.

Matrix metalloproteinase-2 and matrix metalloproteinase-9 codistribute with transcription factors RUNX1/AML1 and ETV5/ERM at the invasive front of endometrial and ovarian carcinoma.

Several matrix metalloproteinases (MMPs) are implicated in the degradation of the epithelial basement membrane (BM), invasiveness, and malignancy of endometrial and ovarian carcinomas. We have recently proposed a cooperative role for RUNX1/AML1 and ETV5/ERM in myometrial infiltration during endometrioid endometrial invasiveness. In the present work, we have characterized the occurrence, levels of expression, and codistribution of gelatinases MMP-2 and -9, and the transcription factors RUNX1/AML1 and ETV5/ERM, together with collagen type IV and laminin chains of the epithelial BM in endometrioid endometrial (EEC) and ovarian endometrioid carcinoma (OEC). MMP-2 and -9 expression levels were up-regulated at the invasive front of both carcinomas, and they showed a relatively high degree of volume codistribution with RUNX1/AML1 and ETV5/ERM. EEC tissue microarrays showed similar significant expression and correlation for MMPs and the transcription factors. When the array samples were grouped according to the carcinoma stages, there was significant correlation in the expression levels for both MMP-2 and -9 with ETV5/ERM. Colocalization of MMP-2 and -9 with epithelial basement membrane component collagen type IV showed close spatial association for both MMPs and discontinuation of collagen type IV expression at the invasive front in both EEC and OEC. BM components laminin α1, α2, α3, α5, and γ2 chains, laminin α5 receptor basal cell adhesion molecule (BCAM), and laminin 332 were all detected both in EEC and OEC. Highest expression levels in EEC were for laminin α3 and in OEC for laminin α5 chain. Laminin γ2 chain and laminin 332 showed discontinuous immunoreactivity in the epithelial basement membrane suggestive of proteolytic degradation. These results indicate concurrent mechanisms in expression of MMP-2 and -9, RUNX1/AML1 and ETV5/ERM, and several of the basement membrane components, which are likely to associate with the invasive stage of EEC and OEC.

Podosome-like structures of non-invasive carcinoma cells are replaced in epithelial-mesenchymal transition by actin comet-embedded invadopodia.

Podosomes and invadopodia are actin-based structures at the ventral cell membrane, which have a role in cell adhesion, migration and invasion. Little is known about the differences and dynamics underlying these structures. We studied podosome-like structures of oral squamous carcinoma cells and invadopodia of their invasive variant that has undergone a spontaneous epithelial-mesenchymal transition (EMT). In 3D imaging, podosomes were relatively large structures that enlarged in time, whereas invadopodia of invasive cells remained small, but were more numerous, degraded more extracellular matrix (ECM) and were morphologically strikingly different from podosomes. In live-cell imaging, highly dynamic, invadopodia-embedded actin tails were frequently released and rocketed through the cytoplasm. Resembling invadopodia, we found new club-ended cell extensions in EMT-experienced cells, which contained actin, cortactin, vinculin and MT1-matrix metalloproteinase. These dynamic cell extensions degraded ECM and, in field emission scanning electron microscopy, protruded from the dorsal cell membrane. Plectin, alphaII-spectrin, talin and focal adhesion kinase immunoreactivities were detected in podosome rings, whereas they were absent from invadopodia. Tensin potentially replaced talin in invadopodia. Integrin alpha(3)beta(1) surrounded both podosomes and invadopodia, whereas integrin alpha(v)beta(5) localized only to invadopodia heads. Pacsin 2, in conjunction with filamin A, was detected early in podosomes, whereas pacsin 2 was not found in invadopodia and filamin A showed delayed accumulation. Fluorescence recovery after photobleaching indicated faster reorganization of actin, cortactin and filamin A in podosomes compared to invadopodia. In conclusion, EMT affects the invasion machinery of oral squamous carcinoma cells. Non-invasive squamous carcinoma cells constitutively organize podosomes, whereas invasive cells form invadopodia. The club-ended cell extensions, or externalized invadopodia, are involved in ECM degradation and maintenance of contact to adhesion substrate and surrounding cells during invasion.

Persistent susceptibility of cathepsin B to irreversible inhibition by nitroxyl (HNO) in the presence of endogenous nitric oxide.

Nitrosation of enzyme regulatory cysteines is one of the key posttranslational modification mechanisms of enzyme function. Frequently such modifications are readily reversible; however, cysteine proteases, such as cathepsin B, have been shown to be covalently and permanently inactivated by nitroxyl (HNO), the one-electron reduction product of NO. Owing to the high reactivity of HNO with NO, endogenous NO production could provide direct protection for the less reactive protein cysteines by scavenging HNO. Additionally, endogenous cellular production of NO could rescue enzyme function by protective nitrosation of cysteines prior to exposure to HNO. Thus, we studied the effect of endogenous NO production, induced by LPS or IFN-gamma, on inhibition of cysteine protease cathepsin B in RAW macrophages. Both LPS and IFN-gamma induce iNOS with generation of nitrate up to 9 muM in the media after a 24-h stimulation, while native RAW 264.7 macrophages neither express iNOS nor generate nitrate. After the 24-h stimulation, the HNO-releasing Angeli's salt (0-316 microM) caused dose-dependent and DTT-irreversible loss of cathepsin B activity, and induction of iNOS activity did not protect the enzyme. The lack of protection was also verified in an in vitro setup, where papain, a close structural analogue of cathepsin B, was inhibited by Angeli's salt (2.7 microM) in the presence of the NO donor DEA/NO (0-316 microM). This clearly showed that a high molar excess of DEA/NO (EC(50) 406 microM) is needed to protect papain from the DTT-irreversible covalent modification by HNO. Our results provide first evidence on a cellular level for the remarkably high sensitivity of active-site cysteines in cysteine proteases for modification by HNO.

Laminin isoforms of lymph nodes and predominant role of alpha5-laminin(s) in adhesion and migration of blood lymphocytes.

During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains and that the same chains, in addition to alpha2, are found in RFs. In functional studies with laminin isoforms covering all Lm alpha chains, alpha5-laminin (Lm-511) was the most adhesion- and migration-promoting isoform for human blood lymphocytes, followed by alpha3- (Lm-332) and alpha4- (Lm-411) laminins, and the lymphocytes used the alpha6beta1 integrin as the primary receptor for the alpha5-laminin. Moreover, Lm-511 strongly co-stimulated T cell proliferation, and blood lymphocytes were able to secrete alpha4- and alpha5-laminins following stimulation. The LN cell number in laminin alpha4-deficient mice compared with wild-type did not differ significantly. This study demonstrates a predominant role for alpha5-laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.

Intracellular co-localization of trypsin-2 and matrix metalloprotease-9: possible proteolytic cascade of trypsin-2, MMP-9 and enterokinase in carcinoma.

Tumor-associated trypsin-2 and matrix metalloprotease-9 (MMP-9) are associated with cancer, particularly with invasive squamous cell carcinomas. They require activation for catalytical competence via proteolytic cascades. One cascade is formed by enterokinase, trypsin-2 and MMP-9; enterokinase activates trypsinogen-2 to trypsin-2, which is an efficient proMMP-9 activator. We describe here that oral squamous cell carcinomas express all members of this cascade: MMP-9, trypsin-2 and enterokinase. The expression of enterokinase in a carcinoma cell line not derived from the duodenum was shown here for the first time. Enterokinase directly cleaved proMMP-9 at the Lys65-Ser66 site, but failed to activate it in vitro. We demonstrated by confocal microscopy that MMP-9 and trypsin-2 co-localized in intracellular vesicles of the carcinoma cells. This co-localization of trypsin-2 and MMP-9 resulted in intracellular proMMP-9 processing that represented fully or partially activated MMP-9. However, although both proteases were present also in various bone tumor tissues, MMP-9 and trypsin-2 never co-localized at the cellular level in these tissues. This suggests that the intracellular vesicular co-localization, storage and possible activation of these proteases may be a unique feature for aggressive epithelial tumors, such as squamous cell carcinomas, but not for tumors of mesenchymal origin.

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas.

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin alpha4, beta1, beta2 and gamma1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin alpha5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin alpha4, beta1, beta2 and gamma1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin alpha5 chain and Lutheran glycoprotein, a receptor for the laminin alpha5 chain. In normal lymphatic capillaries, the presence of primarily alpha4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin alpha5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.

Cooperation of isoforms of laminin-332 and tenascin-CL during early adhesion and spreading of immortalized human corneal epithelial cells.

The repair of corneal wounds requires both epithelial cell adhesion and migration. We have studied the early adhesion process of immortalized human corneal epithelial (HCE) cells and show by field emission scanning electron microscopy (FESEM) that the cells first adhere via foot-like process to the growth substratum and later present lamellar spreading. During early adhesion indirect immunofluorescence showed that the cells codeposited laminin (Lm) -332 and the large subunit of tenascin-C (Tn-CL) as a demarcated plaque beneath the cells. Instead, unprocessed Lm-332 (alpha 3'32) was found in a wider area in cells showing lamellar spreading and was also prominently expressed in the cytoplasm of the migrating marginal cells in the in vitro wounded HCE cultures. Confocal laser scanning microscopy (CLSM) showed that the Golgi apparatus was located to the vicinity of the Lm-332/Tn-CL-containing adhesion plaque and accordingly treatment of the cells with demecolcine, dispersing the Golgi apparatus, prevented the formation of plaques. This suggests that formation of the adhesion plaque depends on a direct vectorial secretion of Lm-332 and Tn-CL to the culture substratum. Instead, cytochalasin B treatment disrupted microfilaments and arborized the cells but did not affect the deposition of Tn-CL/Lm-332 as a plaque beneath the cells. The suggestion was supported by immunoprecipitation experiments which showed that Tn-CL and Lm alpha 3' chain were found in cell-free matrices on the culture substratum of spreading cells but not at all (Tn-CL) or much less (Lm-332) in the culture medium. Quantitative cell adhesion experiments showed that HCE cells did not adhere to plain Tn-C coat and that integrin (Int) alpha(3)beta(1) mediated the adhesion of HCE cells to purified Lm-332 and to Lm-332/Tn-C while Int beta4 did not mediate adhesion to these proteins. Taken together, our data suggest that Lm-332 and Tn-CL cooperate in early adhesion process of HCE cells. Furthermore, the results show that Lm-3'32 isoform functions in the spreading of the cells beyond the early adhesion stage and appears to emerge into HCE cells starting to migrate in experimental wounds.

Snail-dependent and -independent epithelial-mesenchymal transition in oral squamous carcinoma cells.

Disappearance of E-cadherin is a milestone for epithelial-mesenchymal transition (EMT), found both in carcinomas and in some fibrotic diseases. We have studied the mechanisms of EMT in oral squamous cell carcinoma (SCC) cells isolated from primary tumor (43A) and its recurrent tumor (43B). Whereas the cells from primary carcinoma displayed a typical phenotype of squamous epithelial cells including E-cadherin and laminin-332 (laminin-5), cells from recurrent tumor expressed characteristics of dedifferentiated, EMT-experienced tumors. 43B cells expressed E-cadherin repressors ZEB-1/deltaEF1 and especially ZEB-2/SIP1, which therefore appear as candidates for endogenous EMT in these cells. Differences between endogenous and exogenous EMT were assessed by transfecting 43A cells with SNAIL cDNA. SNAIL-transfected cells showed complete EMT phenotype with fibroblastoid appearance, vimentin filaments, E-cadherin/N-cadherin switch, lack of hemidesmosomes and, as a new feature of EMT, lack of laminin-332 synthesis. Upregulation of ZEB-1 and ZEB-2 was evident in these cells, suggesting that SNAIL can regulate these E-cadherin repressors. New monoclonal antibodies against SNAIL showed nuclear immunoreactivity not only in the SNAIL-transfected cells but also in carcinoma cells lacking production of Lm-332 and showing signs of EMT. These results suggest that changes in the epithelial cell differentiation program and EMT in SCC cells can result from the interplay among several E-cadherin repressors; however, SNAIL alone is able to accomplish a complete EMT.

Innervation of the joint and role of neuropeptides.

Rheumatoid arthritis is considered to represent a disease of the synovial membrane, osteoarthritis of the hyaline articular cartilage, and osteoporosis of the bone. It can be questioned to what extent this is true and to what extent these diseases could be considered to be due to extra-articular, extra-skeletal pathology related to the neuroendocrine system. Pain is the main symptom in arthritis. This is related to prostaglandin-mediated sensitization of the primary afferent nociceptive nerves. Accordingly, nonsteroidal anti-inflammatory drugs are used in symptomatic treatment, occasionally together with opioids and tricyclic antidepressants. The midline symmetry and involvement of the richly innervated, small peripheral joints in rheumatoid arthritis have raised speculation about the role of neurogenic inflammation and neuropeptides in its pathogenesis. In contrast to the free nerve endings, the role of the proprioceptive sensors is to provide information of our actual motor performance (the afferent copy of our movements) compared to the efferent motor program, which is activated by our will to move. These include proprioceptors in the skin (e.g., Meissner corpuscles), muscles (annulospiral and flower-spray endings of the muscle spindles), Golgi tendon organs, and Ruffini end organs and Pacinian corpuscles in the superficial and deep layers of the joint capsule. Elderly people may have slow reflexes, lax joints, joint incongruity, and loss of muscle power; obesity, alcohol and medicinal use, and joint pain can be combined with poor/nonexisting capacity for repair and remodeling of the musculoskeletal tissues. Impaired biomechanics contributes to increased joint tenderness, accumulation of minor trauma (secondary osteoarthritis), and falls (osteoporotic fractures). More attention needs to be paid to aging of proprioception, not only to the terminal disease target.

Cathepsin B is a differentiation-resistant target for nitroxyl (HNO) in THP-1 monocyte/macrophages.

We previously showed that the one-electron reduction product of nitric oxide (NO), nitroxyl (HNO), irreversibly inhibits the proteolytic activity of the model cysteine protease papain. This result led us to investigate the differential effects of the nitrogen oxides, such as nitroxyl (HNO), NO, and in situ-generated peroxynitrite on cysteine modification-sensitive cellular proteolytic enzymes. We used Angeli's salt, diethylaminenonoate (DEA/NO), and 3-morpholinosydnoniminehydrochloride (SIN-1), as donors of HNO, NO, and peroxynitrite, respectively. In this study we evaluated their inhibitory activities on the lysosomal mammalian papain homologue cathepsin B and on the cytosolic 26S proteasome in THP-1 monocyte/macrophages after LPS activation or TPA differentiation. HNO-generating Angeli's salt caused a concentration-dependent (62 +/- 4% at 316 muM) inhibition of the 26S proteasome activity, resulting in accumulation of protein-bound polyubiquitinylated proteins in LPS-activated cells, whereas neither DEA/NO nor SIN-1 showed any effect. Angeli's salt, but not DEA/NO or SIN-1, also caused (94 +/- 2% at 316 muM) inhibition of lysosomal cathepsin B activity in LPS-activated cells. Induction of macrophage differentiation did not significantly alter the inhibitory effect of HNO on lysosomal cathepsin B activity, but protected the proteasome from HNO-induced inhibition. The protection awarded by macrophage differentiation was associated with induction of the GSH synthesis rate-limiting enzyme gamma-glutamylcysteine synthetase, as well as with increased intracellular GSH. In conclusion, HNO abrogates both lysosomal and cytosolic proteolysis in THP-1 cells. Macrophage differentiation, associated with upregulation of antioxidant defenses such as increased cellular GSH, does not protect the lysosomal cysteine protease cathepsin B from inhibition.

Effects of two multi-step self-etch primer/adhesives on apoptosis in human gingival fibroblasts in vitro.

Various in vitro studies have shown induction of apoptosis by monomers incorporated to dental restorative materials and adhesive resins, while information regarding the effect of monomer combinations as commercially available products on apoptosis is limited. The aim of this study was to investigate the effects of two multi-step self-etch primer/adhesive systems on apoptosis of cultured primary human gingival fibroblasts. Cells were treated up to 48 h with Clearfil SE Bond (Kuraray, Japan) and FL Bond (Shofu, Japan) at 1:1000 v:v ratio to determine cell proliferation, using 0.02 mM staurosporine as positive control. Apoptosis was assessed using propidium iodide/acridine orange (PI/AO) staining, compared to nontreated controls. When compared to FL Bond, exposure of gingival fibroblasts to Clearfil SE Primer and Clearfil SE Bond resulted in a higher degree of cell proliferation. PI/AO staining revealed typical morphological features of apoptosis in FL Bond and Staurosporine groups, while some cells cultured in the presence of primer and adhesive components of Clearfil SE Bond showed nuclear fragmentation, indicative of early apoptosis. Our results indicate that apoptotic potential of the multi-step self-etch adhesives were material-dependent within the 48 h test period.