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.

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.

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.

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.

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).

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.

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.

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.

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).

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

Human osteoblasts produce cathepsin K.

Healthy bone is a rigid yet living tissue that undergoes continuous remodeling. Osteoclasts resorb bone in the remodeling cycle. They secrete H(+)-ions and proteinases to dissolve bone mineral and degrade organic bone matrix, respectively.

Abnormal distribution of aquaporin-5 in salivary glands in the NOD mouse model for Sjögren's syndrome.

Objective: To localize aquaporin-5 in healthy salivary gland acinar cells and to check if it is abnormally translocated in an experimental NOD mouse model for Sjögren's syndrome (SS).

Methods: Healthy BALB/c control mice and autoimmune focal adenitis NOD mice were studied. Aquaporin-5 was stained using avidin-biotin-peroxidase complex and indirect immunofluorescence staining methods, and visualized using light and laser scanning confocal microscopy.

Interface tissue fibroblasts from loose total hip replacement prosthesis produce receptor activator of nuclear factor-kappaB ligand, osteoprotegerin, and cathepsin K.

Objective: The highly osteolytic interface tissue between the bone and loosening total hip prosthesis is characterized by low pH, formation of foreign body giant cells, osteoclasts, and production of receptor activator of nuclear factor-kappaB (RANKL) and cathepsin K. We hypothesized that fibroblasts in the interface tissue may form a source for RANKL production.

Methods: Primary interface tissue fibroblasts, fibrous joint capsule fibroblasts, and trabecular bone osteoblasts were stimulated with tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-11, or 1alpha,25-(OH)2 vitamin D3.

Pseudosynovial fluid from loosened total hip prosthesis induces osteoclast formation.

Interface tissue between the bone and loosening total hip implant is acidic and highly osteolytic. It is characterized by the formation of cathepsin K positive foreign body giant cells. Similar structures to those found in the normal joint surround the artificial hip joint.

Disease-associated increased HIF-1, alphavbeta3 integrin, and Flt-1 do not suffice to compensate the damage-inducing loss of blood vessels in inflammatory myopathies.

Objective: To analyze the microvascular network in skeletal muscle biopsies from patients with dermatomyositis (DM) and systemic sclerosis (SSc) compared to polymyositis (PM) and systemic lupus erythematosus (SLE), and non-inflammatory myopathies, and to clarify whether reparative angiogenesis-related factors are expressed in parallel to blood vessel damage.

Methods: Immunohistochemical staining of muscle biopsies (10 DM, 10 SSc, 10 PM, 10 SLE, and 10 non-inflammatory myopathies) with antibodies against von Willebrand factor (vWF), hypoxia-inducible factor-1beta (HIF-1beta), beta3 integrin subunit, and vascular endothelial growth factor receptor-1 (VEGFR-1). The TechMate staining robot and biotin-streptavidin protocol were used.