Signaling pathways involved in the regulation of TNFα-induced toll-like receptor 2 expression in human gingival fibroblasts.

Periodontitis is a chronic inflammatory disease characterized by a host inflammatory response against bacteria that leads to destruction of the supporting structures of the teeth. Bacterial components of pathogens in the periodontal pocket are recognized by toll-like receptors (TLRs) that trigger an inflammatory response. In this study, we investigated the effects of the pro-inflammatory cytokine tumor necrosis factor α (TNFα) on TLR2 expression in human gingival fibroblasts. In addition, we examined the signaling pathways involved in the regulation of TNFα-induced TLR2 expression. Our results showed that TNFα increased TLR2 mRNA and protein expression. Microarray analysis and the inhibition of specific signaling pathways demonstrated that c-Jun N-terminal kinases (JNK) and nuclear factor kappa B (NF-κB) were involved in the regulation of TNFα-induced TLR2 expression in gingival fibroblasts. Furthermore, the prostaglandin E(2) (PGE(2)) regulatory enzyme cytosolic phospholipase A(2) (cPLA(2)) and the anti-inflammatory prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), were found to regulate TLR2 mRNA expression stimulated by TNFα. Our findings suggest that these pathways and mediators, through the regulation of TLR2 expression in gingival fibroblasts, may be involved in the pathogenesis of periodontitis. The study provides new insights into the molecular mechanisms underlying the regulation of TLR2, implicated in the chronic inflammatory disease periodontitis.

Expansion and activation kinetics of immune cells during early phase of GVHD in mouse model based on chemotherapy conditioning.

In the present paper, we have investigated early pathophysiological events in graft-versus-host disease (GVHD), a major complication to hematopoietic stem cell transplantation (HSCT). BLLB/c female mice conditioned with busulfan/cyclophosphamide (Bu-Cy) were transplanted with allogeneic male C57BL/6. Control group consisted of syngeneic transplanted Balb/c mice. In allogeneic settings, significant expansion and maturation of donor dendritic cells (DCs) were observed at day +3, while donor T-cells CD8+ were increased at day +5 (230%) compared to syngeneic HSCT. Highest levels of inflammatory cytokines IL-2, IFN-gamma, and TNF-alfa at day +5 matched T-cell activation. Concomitantly naïve T-cells gain effecr-memory phenotype and migrated from spleen to peripheral lymphoid organs. Thus, in the very early phase of GHVD following Bu-Cy conditioning donor, DCs play an important role in the activation of donor T cells. Subsequently, donor naïve T-cells gain effector-memory phenotype and initiate GVHD.

Lentiviral-mediated HoxB4 expression in human embryonic stem cells initiates early hematopoiesis in a dose-dependent manner but does not promote myeloid differentiation.

The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epithelium-cadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developing embryoid bodies. Notably, a high HoxB4 expression in hESCs also seemed to interfere with the formation of germ layers after xenografting into immunodeficient mice. These data suggest that HoxB4-induced effects on hESC-derived HSCs are concentration-dependent during in vitro development and reduce proliferation of other cell types in vitro and in vivo. The application of the transcription factor HoxB4 during early hematopoiesis from hESCs might provide new means for regenerative medicine, allowing efficient differentiation and engraftment of genetically modified hESC clones. Our study highlights the importance of HoxB4 dosage and points to the need for experimental systems allowing controlled gene expression. Disclosure of potential conflicts of interest is found at the end of this article.

Dynamics of fat cell turnover in humans.

Obesity is increasing in an epidemic manner in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. The factors determining fat mass in adult humans are not fully understood, but increased lipid storage in already developed fat cells (adipocytes) is thought to be most important. Here we show that adipocyte number is a major determinant for the fat mass in adults. However, the number of fat cells stays constant in adulthood in lean and obese individuals, even after marked weight loss, indicating that the number of adipocytes is set during childhood and adolescence. To establish the dynamics within the stable population of adipocytes in adults, we have measured adipocyte turnover by analysing the integration of 14C derived from nuclear bomb tests in genomic DNA. Approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index. Neither adipocyte death nor generation rate is altered in early onset obesity, suggesting a tight regulation of fat cell number in this condition during adulthood. The high turnover of adipocytes establishes a new therapeutic target for pharmacological intervention in obesity.

Small heat shock proteins Hsp27 or alphaB-crystallin and the protein components of neurofibrillary tangles: tau and neurofilaments.

The heat-shock proteins (HSPs) Hsp27 and alphaB-crystallin are up-regulated in Alzheimer's disease (AD), but the extent of this and the consequences are still largely unknown. The HSPs are involved in protein degradation and protection against protein aggregation, and they interact with several cytoskeletal components such as microtubules (MT) and neurofilaments (NF). AD pathology includes aggregated proteins (tau, NF), decreased protein degradation, and cytoskeletal disruption. It is thus of interest to investigate more closely the possible roles of the HSPs in AD pathology. The expressions of Hsp27 and alphaB-crystallin in AD brain samples were significantly increased (by approximately 20% and approximately 30%, respectively) and correlated significantly with phosphorylated tau and NF proteins. To investigate the consequences of increased HSP levels on tau and NF regulation, N2a cells were transfected with Hsp27 or alphaB-crystallin constructs, and overexpression of the HSPs was confirmed in the cells. Increased tau phosphorylation at the Ser262 site in the N2a cells was regulated by Hsp27 overexpression (possibly through p70S6k), whereas the overexpression of alphaB-crystallin resulted in decreased levels of phosphorylated tau, NF, and GSK-3beta. It was also shown that overexpression of HSPs causes an increase in the percentage of cells present in the G(1) phase. The results presented suggest that a cellular defense against dysregulated proteins, in the form of Hsp27 and alphaB-crystallin, might contribute to the cell cycle reentry seen in AD cells. Furthermore, Hsp27 might also be involved in AD pathology by aggravating MT disruption by tau phosphorylation.

Differential role of Presenilin-1 and -2 on mitochondrial membrane potential and oxygen consumption in mouse embryonic fibroblasts.

Increasing evidence indicates that mitochondrial alterations contribute to the neuronal death in Alzheimer's disease (AD). Presenilin 1 (PS1) and Presenilin 2 (PS2) mutations have been shown to sensitize cells to apoptosis by mechanisms suggested to involve impaired mitochondrial function. We have previously detected active gamma-secretase complexes in mitochondria. We investigated the impact of PS/gamma-secretase on mitochondrial function using mouse embryonal fibroblasts derived from wild-type, PS1-/-, PS2-/- and PS double knock-out (PSKO) embryos. Measurements of mitochondrial membrane potential (DeltaPsim) showed a higher percentage of fully functional mitochondria in PS1-/- and PSwt as compared to PS2-/- and PSKO cells. This result was evident both in whole cell preparations and in isolated mitochondria. Interestingly, pre-treatment of isolated mitochondria with the gamma-secretase inhibitor L-685,458 resulted in a decreased population of mitochondria with high DeltaPsim in PSwt and PS1-/- cells, indicating that PS2/gamma-secretase activity can modify DeltaPsim. PS2-/- cells showed a significantly lower basal respiratory rate as compared to other cell lines. However, all cell lines demonstrated competent bioenergetic function. These data point toward a specific role of PS2/gamma-secretase activity for proper mitochondrial function and indicate interplay between PS1 and PS2 in mitochondrial functionality.

Myeloablative and immunosuppressive properties of treosulfan in mice.

OBJECTIVE: Treosulfan is a prodrug with a specific clinical activity in ovarian carcinoma and other solid tumors. Due to its myeloablative and immunosuppressive effects, its use in conditioning regimens prior to allogeneic stem cell treatment (SCT) has been proposed. In the present preclinical study, myeloablative as well as immunosuppressive properties of treosulfan were compared with those of busulfan and cyclophosphamide. METHODS: Three groups of BALB/c mice were treated with treosulfan, cyclophosphamide, or busulfan at sublethal doses that maintained survival without bone marrow support. The control group was left untreated. At different intervals, colony-forming unit granulocyte macrophage assay was performed on marrow cells. Additionally, immunological analyses were performed using spleen cells. RESULTS: We found that treosulfan and busulfan induced a high and persisting degree of myeloablation, as compared with cyclophosphamide. Moreover, treosulfan was more effective in depletion of splenic B and T cells in comparison with busulfan and cyclophosphamide. Furthermore, T cells isolated from the spleens of treosulfan- or busulfan-treated mice were not responsive to allogeneic cells compared with that observed in controls and cyclophosphamide-treated mice. Treatment with treosulfan induced only interleukin-2 production in spleen cells for a short time and had no significant effect on synthesis of tumor necrosis factor-alpha and/or interferon-gamma as compared with that observed in splenic T cells isolated from mice treated with either busulfan or cyclophosphamide. CONCLUSION: Our findings suggest that treosulfan possesses both myeloablative and immunosuppressive properties and may be used as a single agent for conditioning prior to bone marrow transplantation.

Effect of altering administration order of busulphan and cyclophosphamide on the myeloablative and immunosuppressive properties of the conditioning regimen in mice.

OBJECTIVE: The present study was designed to investigate the influence of the administration sequence of busulphan (Bu) and cyclophosphamide (Cy) during conditioning regimen on myeloablative and immunosuppressive effects and on engraftment. METHODS: Female Balb/C mice were treated with either Bu-Cy or Cy-Bu (assigned order of administration). Bu was administered as 8.75 mg/kg/day x 4 and Cy as 100 mg/kg/day x 2. The control consisted of untreated animals. Bone marrow and spleen were harvested during the conditioning regimen and for up to 19 days after treatment. Colony-forming unit granulocyte macrophage assay was performed on marrow cells. Immunological analyses were performed using spleen cells. Liver status was determined using aspartate amino transferase (AST), alanine amino transferase (ALT), and bilirubin. Animals assigned for engraftment study were conditioned as above and transplanted using sca-1 cells from male Balb/C donors. Engraftment was followed using fluorescence in situ hybridization up to 30 days posttransplantation. RESULTS: No significant difference in myeloablative effect was observed between treatments. Immunosuppressive activity expressed as CD3+/CD19+ and CD4+/CD8+ was also similar. Levels of cytokines interleukin 2, tumor necrosis factor alpha, and interferon gamma at the end of the conditioning regimen were lower in the Cy-Bu group, while liver enzymes were higher after the Bu-Cy regimen. Engraftment in bone marrow was reached faster within the first 20 days after conditioning with Cy-Bu compared to Bu-Cy. However, no difference in chimerism was observed at 30 days. CONCLUSION: Cy-Bu treatment resulted in lower levels of cytokines, faster bone marrow engraftment, and lower values of liver enzymes compared to Bu-Cy regimen, which may benefit stem cell transplantation outcomes.

Kainic acid-induced excitotoxic hippocampal neurodegeneration in C57BL/6 mice: B cell and T cell subsets may contribute differently to the pathogenesis.

The roles of T cells and B cells in kainic acid (KA)-induced hippocampal lesions were studied in C57BL/6 mice lacking specific T cell populations (CD4, CD8, and CD4/CD8 cells) and B cells [Igh-6(-/-)]. At 48 mg/kg of KA administrated intranasally, KA-induced convulsions were seen in all groups. However, CD4/CD8(-/-) mice exhibited the mildest seizures; the responses of CD8(-/-), Igh-6(-/-) and wild-type mice were intermediate, whereas CD4(-/-) mice displayed much more severe clinical signs and 100% early mortality, indicating that a deficiency of CD4 T cells obviously increased susceptibility to KA-induced brain damage. Histopathological analysis of the mice that survived 7 days after KA administration revealed that CD4/CD8(-/-) mice had the fewest pathologic changes but Igh-6(-/-) mice showed more severe lesions in area CA3 of the hippocampus than CD8(-/-) and wild-type mice. Reactive astrogliosis were prominent in all KA-treated mice. Locomotor activity as assessed by open-field test increased after KA administration in Igh-6(-/-) and wild-type mice only. These results denote the influence of the adaptive immune response on KA-induced hippocampal neurodegeneration and suggest that B cell and T cell subsets may contribute differently to the pathogenesis.