Argonaute 2 sustains the gene expression program driving human monocytic differentiation of acute myeloid leukemia cells.

MicroRNAs are key regulators of many biological processes, including cell differentiation. These small RNAs exert their function assembled in the RNA-induced silencing complexes (RISCs), where members of Argonaute (Ago) family of proteins provide a unique platform for target recognition and gene silencing. Here, by using myeloid cell lines and primary blasts, we show that Ago2 has a key role in human monocytic cell fate determination and in LPS-induced inflammatory response of 1,25-dihydroxyvitamin D3 (D3)-treated myeloid cells. The silencing of Ago2 impairs the D3-dependent miR-17-5p/20a/106a, miR-125b and miR-155 downregulation, the accumulation of their translational targets AML1, VDR and C/EBPß and monocytic cell differentiation. Moreover, we show that Ago2 is recruited on miR-155 host gene promoter and on the upstream region of an overlapping antisense lncRNA, determining their epigenetic silencing, and miR-155 downregulation. These findings highlight Ago2 as a new factor in myeloid cell fate determination in acute myeloid leukemia cells.

Rationalizing the dental curriculum in light of current disease prevalence and patient demand for treatment: form vs. content.

The premise of this paper is that the form and content of dental education do not reinforce each other. What results is suboptimal learning; dissatisfied students; difficulty generating excitement among the brightest to consider careers in dental education; erosion of dentists' self-identity as men and women of science; and doubts over whether dental schools can continue as the primary providers of oral health education. A need for reform exists because dental curricula must be responsive to changes in current and projected disease demographics, to advances in science and technology, and to a changing societal culture affecting patient demand for treatment. Today's dilemma is that dental schools need to continue to graduate competent practitioners to meet present clinical needs while also preparing students for a radically different kind of practice in the future. Possible approaches to resolve this dilemma include: a shift between what constitutes general practice and what constitutes specialty practice; and, the implementation of an asynchronous-distributed model of dental education. Such changes will likely be independently accompanied by changes in the role of universities in society in general that could make feasible many, now-unthinkable, alternative vehicles for providing dental education.

Cutaneous rat wounds express c49a, a novel gene with homology to the human melanoma differentiation associated gene, mda-7.

We have used DD-PCR (differential display-polymerase chain reaction) to identify new genes that are over- or underexpressed during wound repair. DD-PCR performed on excisional wounds identified the expression of rat c49a. Cloning and sequence analysis of the rat c49a gene revealed high homology to a novel human melanoma differentiation associated gene, mda-7. The human mda-7gene isolated from melanoma cell lines, has been linked with human melanoma differentiation, and growth suppression. Moreover, transfection of human mda-7 constructs into human tumor cells suppresses the growth and colony formation of tumor cells from diverse origins. To confirm and relatively quantitate expression of rat c49a gene during repair, specific primer, reduced cycle RT-PCR (reverse transcription-PCR) was performed. RT-PCR showed an approximately 9 to 12-fold elevation of rat c49a mRNA at 12 h to 5 days above nonwounded controls that gradually decreased to approximately 1.5 to 3-fold by day 14. Cloning and sequence analysis of the entire 1200 base pair c49a gene product showed 78% nucleotide homology to human mda-7. Immunohistochemistry studies localized rat C49A expression primarily to fibroblast-like cells at the wound edge and base. The marked up-regulation of rat c49a transcripts during the inflammatory and early granulation tissue phases of wound repair where cellular processes such as re-epithelialization, angiogenesis, and fibroplasia predominate--suggest that c49a is associated with proliferation of fibroblasts in wound healing.

The effect of chitosan (poly-N-acetyl glucosamine) on lingual hemostasis in heparinized rabbits.

PURPOSE: The purpose of this study was to evaluate the effect of chitosan on lingual hemostasis in rabbits whose coagulation pathway had been impaired by administration of intravenous heparin. MATERIALS AND METHODS: Bleeding times were measured for bilateral (15 mm x 2 mm) tongue incisions in 10 New Zealand white rabbits. Using a randomized, blinded experimental design, one incision in each animal was treated with chitosan, and the other was treated with the control vehicle without chitosan. Activated coagulation times and extraoral bleeding times were measured for each animal before, during, and after heparinization. RESULTS: Intravenous infusion of heparin more than tripled the mean activated coagulation time and increased mean systemic bleeding time by 40%. In this heparinized animal model, lingual incisions receiving the experimental substance showed a 43% improvement in bleeding time as compared with lingual incisions receiving the control solution (P< or =.001). Chitosan treatment brought bleeding time of the lingual incision for heparinized animals within the normal range. Scanning electron microscopic evaluation of the incisions treated with chitosan showed an altered red blood cell morphology and an unusual affinity between erythrocytes. CONCLUSIONS: Topical application of chitosan to lingual incisions effectively decreased intraoral bleeding time in a therapeutically anticoagulated (heparinized) rabbit model. Chitosan facilitated lingual hemostasis, possibly through interaction with erythrocytes, linking them together to establish a cellular clot or hemostatic plug.

Human NELL-1 expressed in unilateral coronal synostosis.

Surgical correction of unilateral coronal synostosis offers a unique opportunity to examine the molecular differences between an abnormal and a normal cranial suture. We isolated and identified a cDNA fragment whose expression was up-regulated in the premature fusing and fused coronal sutures, as compared with normal coronal sutures. The nucleotide sequence of the full-length cDNA of this gene, human NELL-1, has approximately 61% homology with the chicken Nel gene. Both chicken Nel and human NELL-1 are comprised of six epidermal growth factor-like repeats. The human NELL-1 messages were localized primarily in the mesenchymal cells and osteoblasts at the osteogenic front, along the parasutural bone margins, and within the condensing mesenchymal cells of newly formed bone in sites of premature sutural fusion. Human multiorgan tissue mRNA blot showed that NELL-1 was specifically expressed in fetal brain but not in fetal kidney, liver, or lung. We also showed that Nell-1 was expressed in rat calvarial osteoprogenitor cells and was largely absent in rat tibiae and fibroblast cell cultures. In conclusion, our data suggest that the NELL-1 gene is preferentially expressed in cranial intramembranous bone and neural tissue (both of neural crest cell origin) and is up-regulated during unilateral premature closure of the coronal suture. The precise role of this gene is unknown.

Expression of apoptosis-associated genes by human dermal scar fibroblasts.

The purpose of this study was to determine if aberrant apoptosis plays a role in pathologic wound healing as manifested by hypertrophic scarring and keloid formation. Apoptosis has recently been found to participate in the transition between granulation tissue and the development of definitive scar. The question that remains to be answered is what stimuli initiate apoptosis during wound healing. Hitherto, regulatory factors and pathways involved have been largely undefined. We investigated heterogeneity among fibroblasts derived from normal skin and keloid scar, by examining apoptotic profiles and pathways for these cells. Quantitative analysis of apoptotic cells using an Annexin-V-FITC binding assay showed that normal skin fibroblast cultures were found to have a two-fold higher percentage of apoptotic cells than did keloid fibroblast cultures. To study apoptotic pathways and related death-associated genes, a ribonuclease protection assay was performed for fibroblasts exposed to anti-Fas antibody and tumor necrosis factor-alpha to activate the Fas/TNF receptor apoptotic pathway. Compared with normal skin fibroblasts, keloid fibroblasts exhibited decreased expression of apoptosis-associated genes.

Effect of TGF-beta 1 on PDGF receptors expression in human scar fibroblasts.

This study examined the effect of exogenous TGF -beta1 on platelet derived growth factor alpha and beta (PDGF-alpha, beta) receptor expression in human dermal fibroblasts derived from both normal cutaneous tissues (normal skin [NSk]) and (normal scar [NSc]) and abnormal scar (keloid). TGF-beta and PDGF are present in the early phases of wound healing and are implicated in tissue fibrosis. In this study, replicate samples of NSk, NSc and keloid fibroblasts were grown to subconfluency in DMEM/10% FBS followed by replacement of media with DMEM/0.1%FBS for 24 hrs. One group of cells (NSk, NSc and keloid) were exposed to 10 ng/mL of exogenous TGF-beta1 for 24 hours, while the other group was used as control with no exposure to exogenous TGF-beta1. RadioImmunoBinding assays, Western and Northern blot analysis were performed to examine both PDGF-alpha and PDGF-beta receptor expression at the transcriptional and post-transcriptional levels. cDNA receptor probes were synthesized using polymerase chain reaction (PCR) with selected primer sets derived from published sequences. Beta-actin probe was used as a control to confirm that the same quantity of RNA was used for each experimental condition. TGF-beta1 was found to upregulate the expression of PDGF-alpha receptor for keloid fibroblasts but not for NSk or NSc fibroblasts. No effect was observed for TGF-beta 1 on PDGF-beta receptor expression for any of the cell lines examined.

Normal human oral keratinocytes are more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine-induced cytotoxicity and apoptosis than HPV-immortalized oral keratinocytes.

Exposure of HPV-immortalized, but not normal human oral keratinocytes, to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) renders the cells tumorigenic. The underlying mechanism of this differential response of normal and immortalized cells was investigated. Normal primary human oral keratinocytes and three HPV-immortalized human oral keratinocyte cell lines exposed to MNNG were evaluated for survival rate, single and double-strand DNA breaks, and the expression of p53 and bcl-2 proteins. MNNG exposure for 2 h induced both greater cytotoxicity and a more rapid kinetic of cell death in normal keratinocytes than in the immortalized cells. Further, normal keratinocytes were more sensitive to lower concentrations of MNNG that were subtoxic for the immortalized cells. Likewise, with lower concentration of MNNG (50 mu M), significant single-strand DNA breaks in normal keratinocytes were induced whereas no such effect was seen in the immortalized cells. Double-strand DNA fragmentation (apoptosis) was observed in normal keratinocytes exposed to 50 mu M MNNG but not in the immortalized cells. Higher concentrations of MNNG (100 mu M) were toxic to both normal and immortalized cells although the normal cells were still more sensitive and with faster kinetics of cell death. MNNG-induced apoptosis was not attributable to down regulation of the anti-apoptotic product bcl-2 in normal cells; however, exposure of normal keratinocytes to MNNG did result in induction of the apoptotic gene p53. No change in p53 level was seen in the immortalized cells. These findings suggest that the selective sensitivity of normal keratinocytes to MNNG-induced apoptosis is in part due to the induction of p53. The HPV-immortalized cells are resistant to MNNG-induced apoptosis and therefore are capable of undergoing mutations affecting cell proliferation and resulting in tumori-genicity.

Autocrine transforming growth factor-beta(1) activity and glycosaminoglycan synthesis by human cutaneous scar fibroblasts.

Transforming growth factor-beta(1) is a well-known and potent biological response modifier that plays an important role in tissue repair and fibrosis. Among the extracellular constituents known to accumulate in fibrotic tissues, glycosaminoglycans are prominent. In this study we examined transforming growth factor-beta(1) synthesis by human dermal fibroblasts derived from both normal and fibrotic cutaneous tissues. We studied the influence of transforming growth factor-beta(1) on glycosaminoglycan synthesis and explored the role of transforming growth factor-beta(1) as an autocrine mediator of its own expression. These investigations are directed at understanding the persistence of the fibrotic phenotype in scarred skin. Transforming growth factor-beta(1) activity was measured by means of a mink lung epithelium growth inhibitory assay. Replicate explants (n = 3) of fibroblasts each derived from normal skin, normal scar, or hypertrophic scar were studied by adding exogenous transforming growth factor-beta(1) at a concentration range of 0 to 10 ng/ml. The resulting conditioned media were removed and assayed for transforming growth factor-beta(1) activity, then the cells were pulsed for an additional 24 hours with radiolabeled glycosaminoglycan precursor, [(3)H]-glucosamine, to evaluate glycosaminoglycan production. Cell-free glycosaminoglycan synthetic profiles were also developed. Transforming growth factor-beta(1) was found to cause a dose-dependent increase in glycosaminoglycan synthesis in hypertrophic scar and normal skin but not in normal scar fibroblasts in cell-mediated glycosaminoglycan synthesis; the reverse was observed in cell-free glycosaminoglycan synthesis, where transforming growth factor-beta(1) increased glycosaminoglycan synthesis in normal scar but not in normal skin or hypertrophic scar. Most endogenous transforming growth factor-beta(1) existed in latent form for normal skin cells but in active form for normal scar and hypertrophic scar fibroblasts.

The role of proteoglycans in hard and soft tissue repair.

Healing of soft and hard tissues results from a progression of events initiated by injury and directed toward reestablishing normal structure and function. The ubiquity of proteoglycans in mammalian tissues virtually guarantees their involvement in tissue restitution. The dramatic advances in cellular and molecular biology in recent years have added significantly to understanding the specific roles played by proteoglycans in wound repair processes.

State of p53, Rb and DCC tumor suppressor genes in human oral cancer cell lines.

The tumor suppressor genes p53, Rb, and DCC were studied in five human oral cancer cell lines (FaDu, SCC-4, HEp-2, 1483, and OEC-M1) and in primary normal human oral keratinocytes (NHOK). All tested cancer lines had similar amount of p53 messages to normal cells, but the cancer lines FaDu and SCC-4 contained significantly higher p53 protein levels than did the normal counterpart. Sequencing p53 cDNA for these cancer cells showed point mutations: In the FaDu cell line, a mutation of CGG to CTG occurred at codon 248; and in the SCC-4 cell line, a mutation of CCC to TCC occurred at codon 151. The HEp-2 and 1483 cancer lines translated very low levels of p53 protein compared to the normal counterpart. Sequencing of p53 cDNA for HEp-2 and 1483 lines showed no mutations. Southern and Northern analyses revealed that these cell lines harbored HPV-18 DNA and expressed the viral E6/E7 protein. The OEC-M1 line showed different restriction fragment length polymorphism for the p53 gene compared with other cells, and did not express p53. All oral cancer cell lines except the OEC-M1 cells expressed both phosphorylated and hypophosphorylated Rb proteins. Further, the OEC-M1 line expressed smaller sized hypophosphorylated Rb proteins compared with normal cells. Unlike the other cancer lines, the HEp-2 and OEC-M1 lines also did not contain DCC mRNAs. These data indicate that "high risk" HPV infections and mutations of p53, Rb, and DCC genes are frequently found in oral cancer cells and may be associated with oral cancer.

CD44 and hyaluronan expression in human cutaneous scar fibroblasts.

Fibrotic disorders of skin and other organs are typically associated with an abnormal accumulation of extracellular matrix. This study focuses on a matrix constituent, hyaluronan-which is known to be altered in fibrotic disorders of skin- and on CD44, a cell adhesion molecule and putative receptor for hyaluronan. Tissue samples were obtained from biopsies of human normal skin, normal cutaneous scar; and hypertrophic cutaneous scar. After culturing, cells were studied by single- and double-labeling immunohistochemistry using the two anti-CD44 monoclonal antibodies, BU-52 and J173, and a biotinylated hyaluronan binding complex probe, b-HABR. Certain cultures were pretreated with Streptomyces hyaluronidase to assess the dependency of CD44 expression on the presence of endogenous hyaluronan. CD44 expression, both in the presence and the absence of exogenous hyaluronan, was quantitated by radioimmunobinding assay. Overall glycosaminoglycan synthesis and identification of hyaluronan were accomplished by precursor incorporation assays and by quantitative cellulose acetate electrophoresis. CD44 was found to be a normal human adult fibroblastic antigen whose expression is markedly increased for hypertrophic scar fibroblasts compared with normal skin fibroblasts. Although hyaluronan was found to be the predominant glycosaminoglycan constituent of the pericellular matrix for these fibroblasts, CD44 attachment to the cell surface is neither mediated by hyaluronan nor is the presence of hyaluronan a prerequisite for CD44 expression. Exogenous hyaluronan induced a decline in measurable CD44 expression for normal skin fibroblasts but not for hypertrophic scar fibroblasts. These observations are compatible with current understanding of the way cells manage the hyaluronan economy of the extracellular matrix and emphasize phenotypic heterogeneities between fibroblasts derived from normal versus scar tissues.

Use of sodium hyaluronate in treating temporomandibular joint disorders: a randomized, double-blind, placebo-controlled clinical trial.

This study assessed the efficacy of high-molecular-weight sodium hyaluronate as a treatment for certain intracapsular temporomandibular joint (TMJ) disorders. One hundred twenty-one patients were studied at three test sites using a randomized, double-blind, placebo-controlled experimental design. Patients were selected on the basis of 1) confirmed diagnosis of either degenerative joint disease (DJD), reducing displaced disc (DDR), or nonreducing displaced disc (DDN); 2) nonresponsiveness to nonsurgical therapies; and 3) severe dysfunction as established by the Helkimo indices (HI), visual analog scales (VASs), and physical measurements of joint movement and joint noise (arthrophonometry [APM]). Subjects received a unilateral upper joint space injection of either 1) 1% sodium hyaluronate in physiologic saline (MedChem Products, Woburn, MA) or 2) USP physiologic saline. Clinical evaluations were performed using HI, VAS, and APM at weekly intervals for the first month and then at monthly intervals up to 6 months postinjection. Statistical analyses for both categorical and continuous variables were performed for each diagnostic category at each examination interval. For DJD, no difference in outcome was seen between treatment groups. For DDN, significant between-group differences were seen through 1 month; however, beyond this time point, the number of DDN patients was insufficient to draw meaningful conclusions concerning efficacy. For DDR, statistically significant within-group and between-group improvement in all three measures (HI, VAS, APM) was seen for the hyaluronate group compared to the saline group throughout the 6-month test period. At the month-2 and month-3 examination intervals, twice as many patients treated with hyaluronate (90%) showed improvement compared to patients given placebo. Further, only 3% of patients with DDR who were treated with hyaluronate relapsed compared with 31% of patients with DDR given placebo.

Binding and internalization of hyaluronate by human cutaneous fibroblasts.

Hyaluronate is a ubiquitous component of mammalian extracellular matrix. It influences numerous cellular processes and accumulates in fibrotic connective tissue disorders. Recently, hyaluronate catabolism has assumed additional importance because of the introduction into clinical practice of therapeutic procedures which deposit high concentrations of hyaluronate directly into tissues. Relatively little is known about the local metabolism, fate, or long-term effects of either endogenous or exogenous hyaluronate at deposition sites. A capacity for degrading hyaluronate within connective tissues, presumably by fibroblasts, has been inferred but remains controversial because direct proof that human fibroblasts endocytose and degrade hyaluronate has been lacking. In the present study, fibroblasts from normal and fibrotic skin were incubated with [3H]-hyaluronate. Binding and internalization of radiolabeled substrate were then measured: Binding assays revealed a saturable, dose-dependent increase in cell surface-associated [3H]-hyaluronate which was enhanced by pretreatment with hyaluronidase. Similar binding curves were obtained for all cells tested. All the cell lines internalized hyaluronate; however, fibroblasts in confluent cultures internalized 3.5- to 4.2-fold more radioactivity per cell than did fibroblasts from corresponding subconfluent cultures (p less than or equal to 0.002). Normal scar fibroblasts showed greater capacity for generating hyaluronate-derived partial degradation products. This work provides clear evidence that human cutaneous fibroblasts are capable of both binding and internalizing hyaluronate, possibly as a prerequisite for degradation.

Effect of chitosan on lingual hemostasis in rabbits with platelet dysfunction induced by epoprostenol.

Chitosan, a complex carbohydrate derivative of shellfish exoskeleton, is shown to enhance lingual hemostasis in rabbits treated with a known antagonist of platelet function, epoprostenol (prostacyclin or PGI2). Bleeding times were measured for bilateral (15 mm x 2 mm) tongue incisions in 10 New Zealand white rabbits. Using a randomized, blinded experimental design, one incision in each animal was treated with chitosan and the other was treated with control vehicle without chitosan. Extraoral bleeding and coagulation times were measured for each animal before, during, and after infusion of epoprostenol. Continuous infusion of epoprostenol increased mean systemic bleeding time 95%. In this platelet dysfunction animal model, lingual incisions receiving the experimental substance showed a 56% improvement in bleeding time in comparison with lingual incisions receiving control solution (P = .003).

Effect of chitosan on lingual hemostasis in rabbits.

Bleeding times were measured for bilateral (15 mm x 2 mm) tongue incisions in 14 New Zealand white rabbits. Using a randomized, blinded experimental design, one incision in each animal was treated with chitosan and the other was treated with control vehicle without chitosan. Extraoral bleeding and coagulation times were also measured for each animal preoperatively, postoperatively, and prior to killing to verify normal bleeding parameters and to evaluate possible systemic effects associated with topical application. Comparison of lingual incisions receiving the experimental substance versus those receiving control solution showed enhanced hemostasis manifested by a 32% (P less than .05) decrease in bleeding time.

General principles of healing pertinent to the periodontal problem.

The healing process involves a series of coordinated procedures initiated by injury and directed toward restoring structural and functional integrity of the disrupted tissues. Wound healing begins immediately after tissue injury occurs and requires close control of degenerative and regenerative processes, which involve numerous cell types and complex interactions between multiple biochemical cascades.