Comparative Evaluation of Different Bone Markers in Peri-implant Crevicular Fluid of Immediate Loaded and Nonloaded Dental Implants.

AIM: The present study was conducted to determine different bone markers in immediate loaded and nonloaded dental implants. MATERIALS AND METHODS: It comprised of 60 patients (males-30, females-30) which were divided into two groups of 30 each. Group I received immediate loaded dental implants, and group II received non-loaded dental implants. Modified bleeding on probing index, peri-implant sulcus depth was assessed in both groups at 1 month, 2 months, 3 months and 4 months. The crevicular fluid was obtained to determine bone markers levels such as transforming growth factor-alpha (TGF-a), osteocalcin (OCN), osteopontin (OPN), parathyroid hormone (PTH) and osteoprotegerin (OPG). RESULTS: Both groups revealed non-significant difference in modified bleeding on probing index and peri-implant sulcus depth (p > 0.05). Bone markers found to be elevated more in group I as compared to group II. However, the difference was non- significant (p > 0.05). CONCLUSION: Transforming growth factor alpha (TGF-a), OCN, OPN, OPG and PTH and parathyroid hormone (PTH) levels were higher in immediate loaded dental implants as compared to nonloaded dental implants. CLINICAL SIGNIFICANCE: Immediate loaded dental implants showed an increase in expression of bone markers such as TNF-a, OCN, OPN, PTH and OPG which may be useful in deciding future of immediate loaded dental implants.

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour.

AIMS: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness. The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. METHODS AND RESULTS: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. CONCLUSIONS: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Drop in transforming growth factor-alpha and osteoprotegerin level in gingival crevicular fluid from patients with gingivitis.

Inflammatory mediators, especially cytokine, play a central role in the pathogenesis of gingivitis. The aim of this study was to identify and quantify the various growth factors, and cytokines in the gingival crevicular fluid (GCF) of patients with gingivitis, as compared with those of control subjects. The levels of cytokine in the samples were determined by their respective ELISAs. The transforming growth factor (TGF)-alpha and osteoprotegerin (OPG) level were significantly lower in patients with gingivitis than in the controls (p < 0.05). Also, there was a positive correlation between TGF-alpha and OPG levels (r = 0.761). These results suggest that the decrease in growth factor TGF-alpha is associated with the pathophysiology and/or the progress of gingivitis.

Common behavioral influences of the ErbB1 ligands transforming growth factor alpha and epiregulin administered to mouse neonates.

Ligands for epidermal growth factor (EGF) receptor (ErbB1), such as EGF, transforming growth factor alpha (TGFalpha), and epiregulin, are enriched in body fluids and blood and regulate development of various peripheral organs. It remains however how such circulating polypeptide growth factors influence brain development and function. Here, we performed peripheral injections of TGFalpha and epiregulin to mouse neonates and evaluated immediate physical and neurochemical development and later behavioral consequences. Subcutaneous administration of TGFalpha and epiregulin increased phosphorylation of brain ErbB1, suggesting their effects on brain development. Repeated their injections similarly enhanced physical development of eyelid opening and tooth eruption during early postnatal stage and resulted in abnormal behavioral traits in the adult stage. Acoustic startle responses of mice treated with these growth factors as neonates were enhanced and prepulse inhibition was decreased without an apparent correlation between prepulse inhibition level and startle intensity. Locomotor activity and fear-learning performance with tone and context cues were not altered, however. These results suggest that circulating ErbB1 ligands in the periphery of neonates have some common influences on later behavioral traits. Abnormal ErbB1 ligand production at neonatal and potentially prenatal stages might therefore associate with neurodevelopmental disorders such as schizophrenia.

TGF3L fusion enhances the antitumor activity of TRAIL by promoting assembly into polymers.

TRAIL, a promising antitumor immuno-agent, exerted limited efficacy in clinical trials. The third disulfide loop of TGF-α (TGF3L peptide) with a very low affinity for EGFR has been reported to enhance the activity of fused antigens or cytokines. We wondered whether fusion of this peptide could enhance TRAIL activity and what the underlying mechanism for this enhancement would be. The TGF3L-TRAIL showed greatly enhanced cytotoxicity in a variety of cancer cell lines while spared normal cells unharmed. Typical apoptosis and cellular caspase activation were potently induced by TGF3L-TRAIL at the concentration levels corresponding to its cytotoxicity. TGF3L-TRAIL was able to activate both DR4 and DR5 the same as TRAIL did. It induced complete cell death in Colo205 through only one receptor when the other one was blocked, different from TRAIL-induced cell death (through DR4 dominantly). TGF3L-TRAIL cytotoxicity was not reduced in some cell lines even if both receptors are blocked simultaneously. Surprisingly, TGF3L-TRAIL self-assembled into stable polymers, which was responsible for its enhanced cytotoxicity. In human tumor xenograft mouse models, TGF3L-TRAIL showed anti-tumor activity similar to or better than TRAIL in different cancer cell types, consistent with its differing enhancement of cytotoxicity in vitro. Taken together, TGF3L fusion of TRAIL obviously enhances the anticancer activity of TRAIL by promoting assembly into polymers, which presents a novel fusion strategy for improving TRAIL function.

Periostin is essential for periodontal ligament remodeling during orthodontic treatment.

Orthodontic tooth movement is a process stimulated and maintained by external tensile stress; periodontal ligament remodeling serves an important role during this process. However, the function and underlying mechanism of periostin (PN) during orthodontic periodontal ligament remodeling remain unclear. The present study established in vitro and in vivo models of orthodontic treatment to investigate the expression levels of PN under conditions of external tensile stress load. These results indicated that tensile stress load increased the expression levels of PN in mouse peridontal ligaments and human periodontal ligament cells (hPDLCs), during orthodontic tooth movement. Furthermore, the present study demonstrated that the expression levels of PN were regulated by transforming grown factor ß, and that PN promotes type I collagen and α­smooth muscle actin expression levels in hPDLCs. Therefore, PN may be essential for periodontal ligament remodeling during orthodontic treatment, and therefore may represent a potential therapeutic target.

Localization and secretion of epidermal growth factor in the parotid gland and its intragastric kinetics in sheep.

Ruminants secrete a large quantity of saliva that is rich in electrolytes; however, it remains unclear whether their parotid saliva contains epidermal growth factor (EGF). The present study was set up to examine the distribution of EGF and transforming growth factor-alpha (TGF-alpha) in the ovine parotid and submandibular glands and the salivary secretion of EGF-like binding activity (EGF-LBA) as the sum of EGF and TGF-alpha in conscious sheep. We also measured changes in the intragastric concentration of EGF-LBA in the ovine rumen and abomasum, and examined the effect of bilateral diversion of parotid saliva on intragastric EGF-LBA concentration in sheep. Both the ovine parotid and, to a lesser extent, the submandibular glands contained EGF-LBA. Immunohistochemical study showed that EGF and TGF-alpha-immunoreactivities were localized in the ductal epithelium in both glands. Transcriptional expression of EGF and TGF-alpha mRNA was demonstrated in both glands by reverse transcription-polymerase chain reaction (RT-PCR). In conscious sheep, the parotid gland continuously secreted EGF-LBA in the saliva before feeding, and the secretion of parotid EGF-LBA was markedly increased during feeding. After diversion of the parotid saliva for 1 week, EGF-LBA concentration in the ruminal fluid, but not in the abomasal fluid, decreased in the postprandial period, indicating that parotid EGF-LBA is a primary source of EGF-LBA for the rumen fluid during the postprandial period in sheep. Moreover, RT-PCR detected the expression of TGF-alpha mRNA in the rumen and abomasum and that of EGF in the abomasum, implying that these stomachs possibly supply, in part, EGF-LBA to the luminal fluid.

Mixture of sugar and povidone--iodine stimulates wound healing by activating keratinocytes and fibroblast functions.

The topical application of a mixture of sugar and povidone--iodine (PI) has been reported to accelerate the healing of cutaneous wounds and ulcers by promoting re-epithelialization and granulation tissue formation as well as having an anti-microbial effect. To clarify the mechanisms accounting for the efficacy of a 70% sugar and 3% PI paste (U-PASTAtrade mark) (SP), various keratinocytes and fibroblasts functions, including proliferation, collagen synthesis, integrin expression, and cytokine and proteinase secretions in the presence of SP were investigated. Cultured human keratinocytes and fibroblasts were treated with various concentrations of SP, SU and PI. The secretion of urokinase-type plasminogen activator (u-PA), transforming growth factor (TGF)-alpha and interleukin-1alpha from keratinocytes, was detected by ELISA. Collagen synthesis of fibroblasts was examined by means of detecting proline uptake. Furthermore, integrin expressions of these cells were analyzed using a flow cytometer. SP and PI increased intra-cellular u-PA of keratinocytes and stimulated the secretion of u-PA and TGF-alpha. Sugar accelerated the extra-cellular u-PA level only. Both SP and sugar increased the collagen synthesis of fibroblasts. SP and PI also remarkably induced the expressions of extra-cellular matrix receptor integrins, alpha1, alpha2, alpha3, alpha4, alpha5 and beta1, on the surface of keratinocytes and fibroblasts. SP, the mixture of sugar and PI, is likely to act on wounds not only as an antibiotic agent, but also as a modulator for keratinocytes and fibroblasts.

EMD in periodontal regenerative surgery modulates cytokine profiles: A randomised controlled clinical trial.

The enamel matrix derivative (EMD) contains hundreds of peptides in different levels of proteolytic processing that may provide a range of biological effects of importance in wound healing. The aim of the present study was to compare the effect of EMD and its fractions on the cytokine profiles from human gingival fibroblasts in vitro and in gingival crevicular fluid (GCF) in a randomized controlled split-mouth clinical study (n = 12). Levels of cytokines in cell culture medium and in GCF were measured by Luminex over a 2-week period. In the clinical study, levels of pro-inflammatory cytokines and chemokines were increased, whereas the levels of transforming growth factor-α (TGF-α) and platelet-derived growth factor-BB (PDGF-BB) were reduced. The in vitro study showed that EMD and its high and low molecular weight fractions reduced the secretion of pro-inflammatory cytokines and chemokines compared to untreated cells. EMD had an effect on levels of cytokines related to fibroplasia, angiogenesis, inflammation and chemotaxis both in vitro and in vivo, however, the anti-inflammatory effect induced by EMD observed in the in vitro study could not be confirmed clinically.

Role of saliva in esophageal defense: implications in patients with nonerosive reflux disease.

BACKGROUND: It has been previously demonstrated that patients with reflux esophagitis exhibit a significant impairment in the secretion of salivary protective components versus controls. However, the secretion of salivary protective factors in patients with nonerosive reflux disease (NERD) is not explored. The authors therefore studied the secretion of salivary volume, pH, bicarbonate, nonbicarbonate glycoconjugate, protein, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α) and prostaglandin E2 in patients with NERD and compared with the corresponding values in controls (CTRL). METHODS: Salivary secretion was collected during basal condition, mastication and intraesophageal mechanical (tubing, balloon) and chemical (initial saline, acid, acid/pepsin, final saline) stimulations, respectively, mimicking the natural gastroesophageal reflux. RESULTS: Salivary volume, protein and TGF-α outputs in patients with NERD were significantly higher than CTRL during intraesophageal mechanical (P < 0.05) and chemical stimulations (P < 0.05). Salivary bicarbonate was significantly higher in NERD than CTRL group during intraesophageal stimulation with both acid/pepsin (P < 0.05) and saline (P < 0.01). Salivary glycoconjugate secretion was significantly higher in the NERD group than the CTRL group during chewing (P < 0.05), mechanical (P < 0.05) and chemical stimulation (P < 0.01). Salivary EGF secretion was higher in patients with NERD during mechanical stimulation (P < 0.05). CONCLUSIONS: Patients with NERD demonstrated a significantly stronger salivary secretory response in terms of volume, bicarbonate, glycoconjugate, protein, EGF and TGF-α than asymptomatic controls. This enhanced salivary esophagoprotection is potentially mediating resistance to the development of endoscopic mucosal changes by gastroesophageal reflux.

Characterization of the synthesis and secretion of transforming growth factor-alpha from salivary glands and saliva.

Whole saliva collected from rat, mouse, and human sources was found to contain high concentrations of transforming growth factor-alpha (TGF alpha) when analyzed by RIA. The concentrations of TGF alpha in unstimulated human saliva (age, 30-45 yr; n = 10; 1.5 +/- 3.1 nM) was reduced with age (age, 55-70 yr; n = 10; 0.4 +/- 0.1 nM), but increased in oral pathologies manifested in xerostomia (age, 57-70; n = 6; 0.8 +/- 0.2 nM) and Paget's disease (age, 58-76; n = 8; 2.0 +/- 0.6 nM). Immunohistochemical localization of TGF alpha in the salivary glands of rats and mice revealed specific immunostaining of the granular ductal cells of the parotid and submandibular glands. Reverse transcription followed by polymerase chain reaction amplification of total RNA from the parotid and submandibular glands of rats and mice demonstrated the presence of TGF alpha mRNA, suggesting endogenous synthesis by the salivary glands. Thus, salivary glands appear to be an exocrine source for a second member of the epidermal growth factor-like growth factor family in the oral cavity.

Feasibility of in vitro culturing of lesional psoriatic keratinocytes in medium containing high calcium concentrations.

Lesional psoriatic keratinocyte (LPK) culture is considered to be difficult under high-Ca2+ conditions in the absence of special proliferative agents. Using a permeable collagen membrane, we obtained a culture of LPKs under high-Ca2+ conditions without any special proliferative agents. Single-cell suspensions were prepared from the epidermis of chronic psoriatic plaques. Cells were inoculated on the collagen membrane suspended slightly above the bottom of a Petri dish. We used a culture medium of Eagle's MEM containing 10% fetal calf serum. LPKs attached to the membrane 12 h after inoculation and gradually spread. They reached a confluent state by the 10th day of culture. We measured the concentrations of TGF alpha and IL-6 in the medium of LPKs, and compared these with the concentrations in normal keratinocyte (NK) cultures. Significantly increased secretion of TGF alpha by LPKs was observed during the initial phase but this secretion subsequently decreased. Concentrations of IL-6 were below the detectable level in both of NKs and LPKs throughout the observation period. Our results demonstrate that cultured LPKs under high-Ca2+ conditions secrete a larger amount of TGF alpha but not IL-6. Our cell culture system, which allows LPKs to spread and stratify, contributes to the study of the pathogenesis of psoriasis.

Effects of retinol on the temporal expression of transforming growth factor-alpha mRNA in the embryonic mouse mandible.

Development of the mouse embryonic mandible from days 9 to 14 involves tissue interactions in the formation of bone, cartilage, salivary glands and teeth. Growth factors may play an important role in these interactions. Epidermal growth factor (EGF) mRNA expression has been characterized and its presence has been shown to be necessary for odontogenesis. In addition, retinol alters the pattern of dental lamina formation; this effect is correlated with an alteration of the expression of the mRNA for this mitogen (EGF). Transforming growth factor-alpha (TGF alpha) mRNA expression has now been characterized by polymerase chain reaction for this entire period of development (days 9-14). Although the mRNA is present at the same time as EGF (days 9 and 10 only), retinol does not alter the expression of this mitogen as it does EGF. This suggests that retinoids may act to control the proliferative pattern of the dental lamina through EGF expression and not TGF alpha expression, although mRNAs for both mitogens are present at the same time.

Functional genomics of oxidant-induced lung injury.

In summary, acute lung injury is a severe (>40% mortality) respiratory disease associated with numerous precipitating factors. Despite extensive research since its initial description over 30 years ago, questions remain about the basic pathophysiological mechanisms and their relationship to therapeutic strategies. Histopathology reveals surfactant disruption, epithelial perturbation and sepsis, either as initiating factors or as secondary complications, which in turn increase the expression of cytokines that sequester and activate inflammatory cells, most notably, neutrophils. Concomitant release of reactive oxygen and nitrogen species subsequently modulates endothelial function. Together these events orchestrate the principal clinical manifestations of the syndrome, pulmonary edema and atelectasis. To better understand the gene-environmental interactions controlling this complex process, we examined the relative sensitivity of inbred mouse strains to acute lung injury induced by ozone, ultrafine PTFE, or fine particulate NiSO4 (0.2 microm MMAD, 15-150 microg/m3). Measuring survival time, protein and neutrophils in bronchoalveolar lavage, lung wet: dry weight, and histology, we found that these responses varied between inbred mouse strains, and susceptibility is heritable. To assess the molecular progression of NiSO4-induced acute lung injury, temporal relationships of 8734 genes and expressed sequence tags were assessed by cDNA microarray analysis. Clustering of co-regulated genes (displaying similar temporal expression patterns) revealed the altered expression of relatively few genes. Enhanced expression occurred mainly in genes associated with oxidative stress, anti-proteolytic function, and repair of the extracellular matrix. Concomitantly, surfactant proteins and Clara cell secretory protein mRNA expression decreased. Genome wide analysis of 307 mice generated from the backcross of resistant B6xA F1 with susceptible A strain identified significant linkage to a region on chromosome 6 (proposed as Aliq4) and suggestive linkages on chromosomes 1, 8, and 12. Combining of these QTLs with two additional possible modifying loci (chromosome 9 and 16) accounted for the difference in survival time noted in the A and B6 parental strains. Combining these findings with those of the microarray analysis has enabled prioritization of candidate genes. These candidates, in turn, can be directed to the lung epithelium in transgenic mice or abated in inducible and constitutive gene-targeted mice. Initial results are encouraging and suggest that several of these mice vary in their susceptibility to oxidant-induced lung injury. Thus, these combined approaches have led to new insights into functional genomics of lung injury and diseases.

Salivary transforming growth factor alpha in patients with Sjögren's syndrome and reflux laryngitis.

INTRODUCTION: Saliva plays a key role in the homeostasis of the digestive tract, through its inorganic components and its protein growth factors. Sjögren's syndrome patients have a higher prevalence of gastroesophageal reflux disease and laryngopharyngeal reflux. Decreased salivary transforming growth factor alpha levels were observed in dyspeptic patients, but there have been no studies in patients with Sjögren's syndrome and laryngopharyngeal reflux. OBJECTIVE: To compare the salivary transforming growth factor alpha levels of patients with Sjögren's syndrome and laryngopharyngeal reflux to those of healthy controls. METHODS: This is a prospective controlled study. Twelve patients with Sjögren's syndrome and laryngopharyngeal reflux and 11 controls were prospectively evaluated. Spontaneous and stimulated saliva samples were obtained to establish salivary transforming growth factor alpha concentrations. RESULTS: The salivary transforming growth factor alpha levels of patients were significantly higher than those of healthy controls. Five patients with laryngopharyngeal reflux also had erosive esophagitis; their salivary transforming growth factor alpha levels were comparable to controls. CONCLUSION: Salivary transforming growth factor alpha level was significantly higher in patients with Sjögren's syndrome and laryngopharyngeal reflux when compared to the control group.

Interaction between IRF6 and TGFA genes contribute to the risk of nonsyndromic cleft lip/palate.

Previous evidence from tooth agenesis studies suggested IRF6 and TGFA interact. Since tooth agenesis is commonly found in individuals with cleft lip/palate (CL/P), we used four large cohorts to evaluate if IRF6 and TGFA interaction contributes to CL/P. Markers within and flanking IRF6 and TGFA genes were tested using Taqman or SYBR green chemistries for case-control analyses in 1,000 Brazilian individuals. We looked for evidence of gene-gene interaction between IRF6 and TGFA by testing if markers associated with CL/P were overtransmitted together in the case-control Brazilian dataset and in the additional family datasets. Genotypes for an additional 142 case-parent trios from South America drawn from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 154 cases from Latvia, and 8,717 individuals from several cohorts were available for replication of tests for interaction. Tgfa and Irf6 expression at critical stages during palatogenesis was analyzed in wild type and Irf6 knockout mice. Markers in and near IRF6 and TGFA were associated with CL/P in the Brazilian cohort (p<10(-6)). IRF6 was also associated with cleft palate (CP) with impaction of permanent teeth (p<10(-6)). Statistical evidence of interaction between IRF6 and TGFA was found in all data sets (p = 0.013 for Brazilians; p = 0.046 for ECLAMC; p = 10(-6) for Latvians, and p = 0.003 for the 8,717 individuals). Tgfa was not expressed in the palatal tissues of Irf6 knockout mice. IRF6 and TGFA contribute to subsets of CL/P with specific dental anomalies. Moreover, this potential IRF6-TGFA interaction may account for as much as 1% to 10% of CL/P cases. The Irf6-knockout model further supports the evidence of IRF6-TGFA interaction found in humans.

Evaluation of wound-healing formulation against sulphur mustard-induced skin injury in mice.

Sulphur mustard (SM) is a bifunctional alkylating agent that causes cutaneous blisters in human and animals. Remedies to SM-induced dermatotoxicity are still in experimental stage. Due to inevitable requirement of a wound-healing formulation against SM-induced skin lesions, efficacy of formulations including povidone iodine, Aloe vera gel, betaine or framycetin sulphate was evaluated in present study. SM was applied percutaneously (5 mg/kg) once on back region of Swiss albino mice; and after 24 hours, DRDE/WH-02 (Defence Research and Development Establishment/ Wound Healant- 02, containing polyvinylpyrrolidone [PVP], A. vera gel and betaine), Ovadine, Soframycin or A. vera gel were applied topically, daily for 3 or 7 days in different groups. Skin sections were subjected to histopathology, histomorphologic grading, tissue leukocytosis, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemistry of inflammatory-reparative biomarkers. DRDE/WH-02 treated mice received highest score on the basis of histomorphologic scale and lowest number of TUNEL-positive cells compared to other groups. DRDE/WH-02 showed better wound healing as evidenced by widespread re-epithelialization, homogenous fibroplasias well supported by the expression of transforming growth factor-α, endothelial nitric oxide synthase (eNOS) and fibroblast growth factor. Upregulation of interleukin 6 in DRDE/WH-02-treated mice skin resulted in increased tissue leukocytosis and an early removal of tissue debris that initiated reparative process at faster rate compared to other groups. In conclusion, DRDE/WH-02 provided better healing effect and can be recommended as an effective wound healant against SM-induced skin injury.

Mesenchymal stromal cells improve salivary function and reduce lymphocytic infiltrates in mice with Sjögren's-like disease.

BACKGROUND: Non-obese diabetic (NOD) mice develop Sjögren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freund's adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45(-)/TER119(-) cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined. METHODOLOGY/PRINCIPAL FINDINGS: Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45(-)/TER119(-) cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3(+) (T(reg)) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-ß), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF). CONCLUSIONS/SIGNIFICANCE: The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

Autocrine-controlled formation and function of tissue-like aggregates by primary hepatocytes in micropatterned hydrogel arrays.

The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyte-produced growth factors and extracellular matrix (ECM). The local concentrations of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol-fibrinogen hydrogels to provide a mechanically compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-α) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of α5ß1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals.

Gene expression of oncogenes, antimicrobial peptides, and cytokines in the development of oral leukoplakia.

OBJECTIVE: The aim of this study was to investigate the expression pattern of oncogenes, antimicrobial peptides, and genes involved in inflammation in leukoplakia of the oral cavity compared with healthy gingiva. STUDY DESIGN: Biopsies of healthy gingiva (n=20) and leukoplakia (n=20), were obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of alpha-defensin (DEFA) 1/3, DEFA-4, S100-A7, deleted-in-oral-cancer (Doc) 1, interleukin (IL) 1beta, IL-6, IL-8, IL-10, tumor necrosis factor (TNF) alpha, cyclooxygenase (Cox) 2, epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor (TGF) beta1, TGF-alpha, collagen-IA1 (Col-1), and tenascin-c were analyzed by real-time reverse-transcription polymerase chain reaction. The proteins encoded by the different genes were visualized by immunostaining. RESULTS: Compared with healthy gingiva (set as 1), there was an increased gene expression of DEFA-4 (179.2-fold), S100-A7 (25.4-fold), EGF (24.8-fold), TGF-beta1 (25.2-fold), and tenascin-c (34.3-fold) in oral leukoplakia. The expression of IL-1beta and Doc-1 was decreased (0.01-fold and 0.2-fold, respectively). CONCLUSIONS: The combination of an increased expression of the antimicrobial peptide DEFA-4, the oncogene S100-A7, EGF, and tenascin-c, and a decreased Doc-1 expression in oral leukoplakia might characterize its potency of malignant transformation. Chronic inflammation seems not to be involved in the development of this lesion.