Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations

BACKGROUND: Transforming growth factors betas (TGF-betas) are implicated in pancreatic tissue repair but their role in acute pancreatitis is not known. To determine whether endogenous TGF-betas modulate the course of caerulein induced acute pancreatitis, caerulein was administered to wild-type (FVB-/-) and transgenic mice that are heterozygous (FVB+/-) for expression of a dominant negative type II TGF-beta receptor. METHODS: After 7 hourly supramaximal injections of caerulein, the pancreas was evaluated histologically and serum was assayed for amylase and lipase levels. Next, the effects of caerulein on amylase secretion were determined in mouse pancreatic acini, and cholecystokinin (CCK) receptor expression was assessed. RESULTS: The normal mouse pancreas was devoid of inflammatory cells whereas the pancreas from transgenic mice contained lymphocytic infiltrates. Caerulein injection in wild-type mice resulted in 6- and 36-fold increases in serum amylase and lipase levels, respectively, increased serum trypsinogen activation peptide (TAP) levels, gross oedema and a marked inflammatory response in the pancreas that consisted mainly of neutrophils and macrophages. By contrast, FVB+/- mice exhibited minimal alterations in response to caerulein with attenuated neutrophil-macrophage infiltrates. Moreover, acini from FVB+/- mice did not exhibit restricted stimulation at high caerulein concentrations, even though CCK receptor mRNA levels were not decreased. CONCLUSION: Our findings indicate that a functional TGF-beta signalling pathway may be required for caerulein to induce acute pancreatitis and for the CCK receptor to induce acinar cell damage at high ligand concentrations. Our results also support the concept that restricted stimulation at high caerulein concentrations contributes to the ability of caerulein to induce acute pancreatitis.

The etiology of liver damage imparts cytokines transforming growth factor beta1 or interleukin-13 as driving forces in fibrogenesis

It is unknown whether transforming growth factor beta1 (TGF-beta1) signaling uniformly participates in fibrogenic chronic liver diseases, irrespective of the underlying origin, or if other cytokines such as interleukin (IL)-13 share in fibrogenesis (e.g., due to regulatory effects on type I pro-collagen expression). TGF-beta1 signaling events were scored in 396 liver tissue samples from patients with diverse chronic liver diseases, including hepatitis B virus (HBV), hepatitis C virus (HCV), Schistosoma japonicum infection, and steatosis/steatohepatitis. Phospho-Smad2 staining correlated significantly with fibrotic stage in patients with HBV infection (n = 112, P < 0.001) and steatosis/steatohepatitis (n = 120, P < 0.01), but not in patients with HCV infection (n = 77, P > 0.05). In tissue with HBx protein expression, phospho-Smad2 was detectable, suggesting a functional link between viral protein expression and TGF-beta1 signaling. For IL-13, immunostaining correlated with fibrotic stage in patients with HCV infection and steatosis/steatohepatitis. IL-13 protein was more abundant in liver tissue lysates from three HCV patients compared with controls, as were IL-13 serum levels in 68 patients with chronic HCV infection compared with 20 healthy volunteers (72.87 +/- 26.38 versus 45.41 +/- 3.73, P < 0.001). Immunohistochemistry results suggest that IL-13-mediated liver fibrogenesis may take place in the absence of phospho-signal transducer and activator of transcription protein 6 signaling. In a subgroup of patients with advanced liver fibrosis (stage > or =3), neither TGF-beta nor IL-13 signaling was detectable. Conclusion: Depending on the cause of liver damage, a predominance of TGF-beta or IL-13 signaling is found. TGF-beta1 predominance is detected in HBV-related liver fibrogenesis and IL-13 predominance in chronic HCV infection. In some instances, the underlying fibrogenic mediator remains enigmatic.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

Attenuation of fibrosis in vitro and in vivo with SPARC siRNA.

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.

Characterization and mechanism of action of suppressor factors derived from human T cell hybridomas

This laboratory developed human T-cell hybridomas which constitutively secrete suppressor factors (SF) capable of inhibiting immune responses (Hybridoma 6:589 (1987). The mechanisms by which human T-cell hybridoma-derived SFs (designated 160 and 169) and Jurkat leukemic T-cell line derived SF inhibit the proliferative response to mitogen by human PBMC were investigated. The Jurkat SF had a pI of 5.2 whereas the 160 and 169 SF had pI of 5.7 and 4.7 (two peaks) and 4.7, respectively. The SF was not transforming growth factor-beta based upon neutralization and iummunoprecipitation experiments with anti-TGF-beta polyclonal antibody. Il-2 production by human PBMC cultured with Con A or OKT3 mAb in the presence of SF was found to be inhibited by greater than 80%. The proliferative responses of SF treated PBMC could not be restored by addition of exogeneous human IL-2. Inhibition of the proliferative responses could not be reversed by addition of exogenous rIL-1, rIL-2 or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on Con A activated cultures time points was not affected by treatment with any SFs. Both the 160 and 169 hybridoma-derived SFs were found to arrest PHA induced cell cycle progression in G$\sb0$/G$\sb1$ phase, whereas SF from the Jurkat T-cell line arrested progression in the S phase. Pretreatment of PBMC with SF prior to the addition of mitogen, followed by washing, did not alter the proliferative response of these PBMC nor their cell cycle progression suggesting that cell activation is necessary for these SF to inhibit proliferative responses. Northern blot analysis of total mRNA from mitogen stimulated PBMC in the presence of SF, revealed a time dependent accumulation of an IL-2 specific mRNA of increased size (2.8 kB) in addition to the expected 1.0 kB mature IL-2 message. Interferon-gamma mRNA was of the appropriate size but its half-life was prolonged in SF treated cultures. IL-2 receptor and IL-1 beta mRNA expression was not altered in these cells. ^

Chromosomal organization and evolutionary conservation of the human chromosome 19q linkage group containing three DNA repair genes

A series of human-rodent somatic cell hybrids were investigated by Southern blot analysis for the presence or absence of twenty-six molecular markers and three isozyme loci from human chromosome 19. Based on the co-retention of these markers in the various independent hybrid clones containing portions of human chromosome 19 and on pulsed field mapping, chromosome 19 is divided into twenty ordered regions. The most likely marker order for the chromosome is: (LDLR, C3)-(cen-MANNB)-D19S7-PEPD-D19S9-GPI-TGF$ \beta$-(CYP2A, NCA, CGM2, BCKAD)-PSG1a-(D19S8, XRCC1)-(D19S19, ATP1A3)-(D19S37, APOC2)-CKMM-ERCC2-ERCC1-(D19S62, D19S51)-D19S6-D19S50-D19S22-(CGB, FTL)-qter.^ The region of 19q between the proximal marker D19S7 and the distal gene coding for the beta subunit of chorionic gonadotropin (CGB) is about 37 Mb in size and covers about 37 cM genetic distance. The ration of genetic to physical distance on 19q is therefore very close to the genomic average OF 1 cM/Mb. Estimates of physical distances for intervals between chromosome 19 markers were calculated using a mapping function which estimates distances based on the number of breaks in hybrid clone panels. The consensus genetic distances between individual markers (established at HBM10) were compared to these estimates of physical distances. The close agreement between the two estimates suggested that spontaneously broken hybrids are as appropriate for this type of study as radiation hybrids.^ All three DNA repair genes located on chromosome 19 were found to have homologues on Chinese hamster chromosome 9, which is hemizygous in CHO cells, providing an explanation for the apparent ease with which mutations at these loci were identified in CHO cells. Homologues of CKMM and TGF$\beta$ (from human chromosome 19q) and a mini-satellite DNA specific to the distal region of human chromosome 19q were also mapped to Chinese hamster 9. Markers from 19p did not map to this hamster chromosome. Thus the q-arm of chromosome 19, at least between the genes PEPD and ERCC1, appears to be a linkage group which is conserved intact between humans and Chinese hamsters. ^

Analysis of the mechanisms that maintain coordinate production of $\alpha$- and $\beta$-tubulin in mammalian cells

Alpha and beta tubulin are essential proteins in all eukaryotic cells. To study how cells maintain coordinate levels of these two interacting proteins, we have used PCR to add a 9 amino acid epitope from influenza hemagglutinin protein onto the carboxyl terminus of $\alpha$1 and $\beta$1-tubulin. The chimeric tubulin genes (HA$\alpha$1 and HA$\beta$1) were transfected into CHO cells and cell lines that stably express each gene were selected. Cells transfected with HA-tubulin do not exhibit any gross changes in growth or morphology. Immunofluorescence analysis demonstrated that HA-tubulins incorporate into both cytoplasmic and spindle microtubules. A quantitative biochemical assay was used to show that HA-tubulins incorporate into microtubules to a normal extent and do not alter the steady state distribution of endogenous tubulin between monomer and polymer pools. Two-dimensional gel analysis of pulse-labeled cells indicated that when HA$\beta$1-tubulin is expressed at high levels, it slightly represses the synthesis of the endogenous $\beta$-tubulin but produces a small increase in the synthesis of $\alpha$-tubulin. Analysis of cells labeled to steady state showed that HA$\beta$1-tubulin accumulates to a similar level as the wild-type gene product, but together these polypeptides produce only a small increase in total tubulin content consistent with the increased synthesis of $\alpha$-tubulin. It thus appears that HA$\beta$1-tubulin successfully competes with endogenous $\beta$-tubulin for heterodimer formation and that free $\beta$-tubulin subunits (endogenous and HA$\beta$1) are selectively degraded to maintain coordinate amounts of $\alpha$- and $\beta$-tubulin. In addition, the increased synthesis of $\alpha$-tubulin suggested the existence of a mechanism to ensure coordinate synthesis of $\alpha$- and $\beta$-tubulin subunits. To analyze whether reciprocal changes in endogenous tubulin synthesis occur when $\alpha$-tubulin is overexpressed, stably transfected CHO cell lines were isolated in which HA$\alpha$1-tubulin represents 50% of the total $\alpha$-tubulin, and its relative abundance can be further increased to 85-90% by treatment with sodium butyrate. In contrast with results obtained using HA$\beta$1-tubulin, transfection of HA$\alpha$1-tubulin decreased the synthesis of endogenous $\alpha$-tubulin to 60% of normal with little or no change in $\beta$-tubulin synthesis. When the transfected cells were treated with sodium butyrate to further increase HA$\beta$1-tubulin production, a larger decrease in the synthesis of endogenous $\alpha$-tubulin (to 30% of normal) was observed. The repression on the synthesis of endogenous $\alpha$-tubulin polypeptide was found to be directly proportional to the expression of HA$\alpha$1-tubulin indicating the existence of an autoregulatory loop, where $\alpha$-tubulin inhibits its own synthesis. To determine whether overproduction of HA$\alpha$1-tubulin affected the transcription, message stability or translation of endogenous $\alpha$-tubulin, the steady state levels of $\alpha$-tubulin mRNA were analyzed by ribonuclease protection assays. The results showed that the steady state level of $\alpha$-tubulin mRNA is not affected by the overexpression of HA$\alpha$1-tubulin, indicating that the repression is translational. The results are compatible with a model in which $\beta$-tubulin synthesis is largely unperturbed by overexpression of other tubulin subunits, and excess $\beta$-tubulin subunits are rapidly degraded to maintain coordinate $\alpha$- and $\beta$-tubulin levels at steady state. In contrast, free $\alpha$-tubulin represses its own synthesis at the translational level, suggesting that its level of production may be controlled by the amount of $\beta$-tubulin available for heterodimer formation. ^

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

drp, A novel cell density regulated protein

Growing cells are continuously processing signals of all varieties and responding to these signals by changes in cellular gene expression. One signal that cells in close proximity relay to each other is cell-cell contact. Non-transformed cells respond to cell-cell contact by arrest of growth and entry into G$\sb0,$ a process known as contact inhibition. Transformed cells do not respond to contact inhibition and continue to grow to high cell density, forming foci when in cell culture and tumors in the living organism. The events surrounding the generation, transduction, and response to cellular contact are poorly understood. In the present study, a novel gene product, drp, is shown to be expressed at high levels in cultured cells at high cell density. This density regulated protein, drp, has an apparent molecular weight of 70 kDa. Northern analysis shows drp to be highly expressed in cardiac and skeletal muscle and least abundant in lung and kidney tissues. By homology to two independently derived sequence tagged sites (STSs) used in the human genome project, drp or a closely related sequence maps to human chromosome 12. Density-dependent increases in drp expression have been demonstrated in six different cell lines including NIH 3T3, Hela and a human teratocarcinoma cell line, PA-1. Cells exhibit increased drp expression both when they are plated at increasing concentrations per unit area, or plated at low density and allowed to grow naturally to higher cell density. Cells at high density can exhibit several phenotypes including growth arrest, accumulation of soluble factors in the media, and increased numbers of cell contacts. Growth arrest by serum starvation or TGF-$\beta$ treatment fails to produce an increase in drp expression. Similarly, treatment of low density cells with conditioned media from high density cells fails to elicit drp expression. These results argue that neither soluble factors accumulated or expressed at high density nor simple exit from the cell cycle is sufficient to produce an increase in drp expression. The expression of drp appears to be uniquely regulated by cell density alone. ^

Fibrillins: Sequence, phylogeny and carboxy terminal domain proteolytic processing

Fibrillin-1 and -2 are large secreted glycoproteins that are known to be components of extracellular matrix microfibrils located in the vasculature, basement membrane and various connective tissues. These microfibrils are often associated with a superstructure known as the elastic fiber. During the development of elastic tissues, fibrillin microfibrils precede the appearance of elastin and may provide a scaffolding for the deposition and crosslinking of elastin. Using RT/PCR, we cloned and sequenced 3.85Kbp of the FBN2 gene. Five differences were found between our contig sequence and that published by Zhang et al. (1995). Like many extracellular matrix proteins, the fibrillins are modular proteins. We compared analogous domains of the two fibrillins and also members of the latent TGF-$\beta$ binding protein (LTBP) family to determine their phylogenetic relationship. We found that the two families are homologous. LTBP-2 is the most similar to the fibrillin family while FBN-1 is the most similar to the LTBP family. The fibrillin-1 carboxy terminal domain is proteolytically processed. Two eukaryotic protein expression systems, baculoviral and CHO-K1, were developed to examine the proteolytic processing of the carboxy terminal domain of the fibrillin-1 protein. Both expression systems successfully processed the domain and both processed a mutant less efficiently. In the CHO-K1 cells, processing occurred intracellularly. ^

Long-term synaptic facilitation of tail sensorimotor connections in {\it Aplysia\/}

Long-term sensitization in Aplysia is a well studied model for the examination of the cellular and molecules mechanisms of long-term memory. Several lines of evidence suggest long-term sensitization is mediated at least partially by long-term synaptic facilitation between the sensory and motor neurons. The sensitization training and one of its analogues, serotonin (5-HT), can induce long-term facilitation. In this study, another analogue to long-term sensitization training has been developed. Stimulation of peripheral nerves of pleural-pedal ganglia preparation induced long-term facilitation at both 24 hr and 48 hr. This is the first report that long-term facilitation in Aplysia persists for more than 24 hr, which is consistent with the observation that long-term sensitization lasts for more than one day. Thus, the data support the hypothesis that long-term facilitation is an important mechanism for long-term sensitization.^ One of the major differences between short-term and long-term facilitation is that long-term facilitation requires protein synthesis. Therefore, the effects of anisomycin, a protein synthesis inhibitor, on long-term facilitation was examined. Long-term facilitation induced by nerve stimulation was inhibited by 2 $\mu$M anisomycin, which inhibits $\sim$90% of protein synthesis. Nevertheless, at higher concentration (20 $\mu$M), anisomycin induced long-term facilitation by itself, which raises an interesting question about the function of anisomycin other than protein synthesis inhibition.^ Since protein synthesis is critical for long-term facilitation, a major goal is to identify and functionally characterize the molecules whose mRNA levels are altered during the formation of long-term facilitation. Behavioral training or its analogues (nerve stimulation and 5-HT) increases the level of mRNA of calmodulin (CaM). Thus, the role of Ca$\sp{2+}$-CaM-dependent protein kinase II (CaMKII), a major substrate of CaM, in long-term facilitation induced by nerve stimulation was examined. KN-62, a specific CaMKII inhibitor, did not block either the induction or the maintenance of long-term facilitation induced by nerve stimulation. These data indicate that CaMKII may not be involved in long-term facilitation. Another protein whose mRNA level of a molecule was increased by the behavioral training and the treatment of 5-HT is Aplysia tolloid/BMP-1-like protein 1 (apTBL-1). Tolloid in Drosophila and BMP-1 in human tissues are believed to be secreted as a metalloprotease to activate TGF-$\beta.$ Thus, the long-term effects of recombinant human TGF-$\beta1$ on synaptic strength were examined. Treatment of ganglia with TGF-$\beta1$ produced long-term facilitation, but not short-term or intermediate-term facilitation ($\le$4 hr). In addition, TGF-$\beta1$ and 5-HT were not additive in producing long-term facilitation, which indicates an interaction between two cascades. Moreover, 5-HT-induced facilitation (at both 24 hr and 48 hr) and nerve stimulation-induced facilitation (at 24 hr) were inhibited by TGF-$\beta$ sRII, a TGF-$\beta$ inhibitor. These results suggest that TGF-$\beta$ is part of the cascade of events underlying long-term sensitization, and also indicate that a signaling molecule used in development may also have functions in adult neuronal plasticity. ^

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Nuclear Factor I-C acts as a regulator of hepatocyte proliferation at the onset of liver regeneration

BACKGROUND & AIMS: Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed abnormal skin wound healing and growth of its appendages, suggesting a role in controlling cell proliferation in adult regenerative processes. Liver regeneration following partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes lead to their rapid and phased proliferation. Although NFI-C is highly expressed in the liver, no hepatic function was yet established for this transcription factor. This study aimed to determine whether NFI-C may play a role in hepatocyte proliferation and liver regeneration. METHODS: Liver regeneration and cell proliferation pathways following two-thirds PH were investigated in NFI-C knockout (ko) and wild-type (wt) mice. RESULTS: We show that the absence of NFI-C impaired hepatocyte proliferation because of plasminogen activator I (PAI-1) overexpression and the subsequent suppression of urokinase plasminogen activator (uPA) activity and hepatocyte growth factor (HGF) signalling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wt mice. The subsequent transient down regulation of NFI-C, as can be explained by a self-regulatory feedback loop with transforming growth factor beta 1 (TGF-ß1), may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. CONCLUSION: NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration.

Conditioned Medium of Demineralized Freeze-Dried Bone Activates Gene Expression in Periodontal Fibroblasts in Vitro.

BACKGROUND: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-β signaling in mesenchymal cells. The aim of the study was to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium (DBCM). Changes in the expression of TGF-β target genes were determined. RESULTS: DBCM altered the expression of TGF-β target genes, e.g., adrenomedullin, pentraxin 3, KN Motif And Ankyrin Repeat Domains 4, interleukin 11, NADPH oxidase 4, and BTB (POZ) Domain Containing 11, by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-β receptor type I kinase inhibitor SB431542, but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-β target genes. CONCLUSION: The findings suggest that the conditioned medium from demineralized bone matrix can activate TGF-β signaling in oral fibroblasts. KEYWORDS: TGF-beta superfamily proteins; bone; bone substitutes; bone transplantation; conditioned media; freeze drying

Bone-Conditioned Medium Changes Gene Expression in Bone-Derived Fibroblasts.

PURPOSE Autologous bone is used for augmentation in the course of oral implant placement. Bone grafts release paracrine signals that can modulate mesenchymal cell differentiation in vitro. The detailed genetic response of the bone-derived fibroblasts to these paracrine signals has remained elusive. Paracrine signals accumulate in bone-conditioned medium (BCM) prepared from porcine cortical bone chips. MATERIALS AND METHODS In this study, bone-derived fibroblasts were exposed to BCM followed by a whole genome expression profiling and downstream quantitative reverse transciptase polymerase chain reaction of the most strongly regulated genes. RESULTS The data show that ADM, IL11, IL33, NOX4, PRG4, and PTX3 were differentially expressed in response to BCM in bone-derived fibroblasts. The transforming growth factor beta (TGF-β) receptor 1 antagonist SB431542 blocked the effect of BCM on the expression of the gene panel, except for IL33. CONCLUSION These in vitro results extend existing evidence that cortical bone chips release paracrine signals that provoke a robust genetic response in mesenchymal cells that is not exclusively mediated via the TGF-β receptor. The present data provide further insights into the process of graft consolidation.