The anti-epileptic drug valproic acid causes malformations in the developing craniofacial skeleton of zebrafish larvae.

Valproic acid (VPA) is an anti-epileptic drug known to cause congenital craniofacial abnormalities, including orofacial clefts (OFC). The exact mechanisms by which VPA leads to craniofacial skeletal malformations are poorly understood. In this study, we investigated the effects of VPA on cartilage and bone formation in the zebrafish larval head during 1-13 hpf (early) and 25-37 hpf (late) development in which cranial neural crest cells (CNCCs) arise and then proliferate and differentiate, respectively. Double-staining for cartilage and bone at 5 dpf revealed that VPA reduced cartilage and bone formation in a dose-dependent manner after both early or late exposure. Several different CNCC-derived cartilage and bone elements were affected in both groups. In the early group (100 µM VPA), the posterior head length and the ethmoid plate were reduced in length (both p < 0.01), while mineralization of 4 out of 9 bone elements was often lacking (all p < 0.01). In the late group (100 µM VPA), also the posterior head length was reduced as well as the length of the ceratohyals (both p < 0.01). Similar to early exposure, mineralization of 3 out of 9 bone elements was often lacking (all p < 0.01). These results indicate that both CNCC formation (early) and differentiation (late) are hampered by VPA treatment, of which the consequences for bone and cartilage formation are persistent at 5 dpf. Indeed, we also found that the expression of several genes related to cartilage and bone was upregulated at 5 dpf. These data indicate a compensatory reaction to the lack of cartilage and bone. Altogether, VPA seems to induce craniofacial malformations via disturbed CNCC function leading to defects in cartilage and bone formation.

Orofacial Muscles: Embryonic Development and Regeneration after Injury.

Orofacial congenital defects such as cleft lip and/or palate are associated with impaired muscle regeneration and fibrosis after surgery. Also, other orofacial reconstructions or trauma may end up in defective muscle regeneration and fibrosis. The aim of this review is to discuss current knowledge on the development and regeneration of orofacial muscles in comparison to trunk and limb muscles. The orofacial muscles include the tongue muscles and the branchiomeric muscles in the lower face. Their main functions are chewing, swallowing, and speech. All orofacial muscles originate from the mesoderm of the pharyngeal arches under the control of cranial neural crest cells. Research in vertebrate models indicates that the molecular regulation of orofacial muscle development is different from that of trunk and limb muscles. In addition, the regenerative ability of orofacial muscles is lower, and they develop more fibrosis than other skeletal muscles. Therefore, specific approaches need to be developed to stimulate orofacial muscle regeneration. Regeneration may be stimulated by growth factors such fibroblast growth factors and hepatocyte growth factor, while fibrosis may be reduced by targeting the transforming growth factor ß1 (TGFß1)/myofibroblast axis. New approaches that combine these 2 aspects will improve the surgical treatment of orofacial muscle defects.

Fgf8a mutation affects craniofacial development and skeletal gene expression in zebrafish larvae.

Craniofacial development is tightly regulated and therefore highly vulnerable to disturbance by genetic and environmental factors. Fibroblast growth factors (FGFs) direct migration, proliferation and survival of cranial neural crest cells (CNCCs) forming the human face. In this study, we analyzed bone and cartilage formation in the head of five dpf fgf8ati282 zebrafish larvae and assessed gene expression levels for 11 genes involved in these processes. In addition, in situ hybridization was performed on 8 and 24 hours post fertilization (hpf) larvae (fgf8a, dlx2a, runx2a, col2a1a). A significant size reduction of eight out of nine craniofacial cartilage structures was found in homozygous mutant (6-36%, P<0.01) and heterozygous (7-24%, P<0.01) larvae. Also, nine mineralized structures were not observed in all or part of the homozygous (0-71%, P<0.0001) and heterozygous (33-100%, P<0.0001) larvae. In homozygote mutants, runx2a and sp7 expression was upregulated compared to wild type, presumably to compensate for the reduced bone formation. Decreased col9a1b expression may compromise cartilage formation. Upregulated dlx2a in homozygotes indicates impaired CNCC function. Dlx2a expression was reduced in the first and second stream of CNCCs in homozygous mutants at 24 hpf, as shown by in situ hybridization. This indicates an impairment of CNCC migration and survival by fgf8 mutation.

Acquired cancer resistance to combination immunotherapy from transcriptional loss of class I HLA.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor-immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.

Neonatal Satellite Cells Form Small Myotubes In Vitro.

Although palatal muscle reconstruction in patients with cleft palate takes place during early childhood, normal speech development is often not achieved. We hypothesized that the intrinsic properties of head satellite cells (SCs) and the young age of these patients contribute to the poor muscle regeneration after surgery. First, we studied the fiber type distribution and the expression of SC markers in ex vivo muscle tissue from head (branchiomeric) and limb (somite-derived) muscles from neonatal (2-wk-old) and young (9-wk-old) rats. Next, we cultured SCs isolated from these muscles for 5, 7, and 9 d, and investigated the in vitro expression of SC markers, as well as changes in proliferation, early differentiation, and fusion index (myotube formation) in these cells. In our ex vivo samples, we found that virtually all myofibers in both the masseter (Mass) and the levator veli palatini (LVP) muscles contained fast myosin heavy chain (MyHC), and a small percentage of digastric (Dig) and extensor digitorum longus myofibers also contained slow MyHC. This was independent of age. More SCs were found in muscles from neonatal rats as compared with young rats [17.6 (3.8%) v. 2.3 (1.6%); P < 0.0001]. In vitro, young branchiomeric head muscle (BrHM) SCs proliferated longer and differentiated later than limb muscle SCs. No differences were found between SC cultures from the different BrHMs. SC cultures from neonatal muscles showed a much higher proliferation index than those from young animals at 5 d (0.8 v. 0.2; P < 0.001). In contrast, the fusion index in neonate SCs was about twice as low as that in SCs from young muscles at 9 d [27.6 (1.4) v. 62.8 (10.2), P < 0.0001]. In conclusion, SCs from BrHM differ from limb muscles especially in their delayed differentiation. SCs from neonatal muscles form myotubes less efficiently than those from young muscles. These age-dependent differences in stem cell properties urge careful consideration for future clinical applications in patients with cleft palate.

Rate of orthodontic tooth movement after changing the force magnitude: an experimental study in beagle dogs.

OBJECTIVES: To study a possible dose-response relation between force magnitude and rate of orthodontic tooth movement by altering forces during bodily orthodontic tooth movement. SETTING AND SAMPLE POPULATION: Eight young adult beagle dogs were used. The experiments were carried out in the Central Animal Facility, and all analyses were conducted in the Department of Orthodontics and Oral Biology, Radboud University Nijmegen Medical Centre. MATERIALS AND METHODS: Orthodontic appliances were placed exerting a reciprocal force on the mandibular second premolars and first molars. A force of 10 or 300 cN was randomly assigned to each side of the dogs. After 22 weeks, all forces were changed to 600 cN. Based on intra-oral measurements, tooth movement rates were calculated. RESULTS: The premolars showed no difference in the rates of tooth movement with 10 or 300 cN. Replacing 10 for 600 cN increased the rate, but replacing 300 for 600 cN did not. Molars moved faster with 300 than with 10 cN, and changing both forces to 600 cN increased the rate of tooth movement. Data from all teeth were pooled considering their relative root surfaces, and a logarithmic relation was found between force and rate of tooth movement. CONCLUSIONS: Only in the very low force range, a positive dose-response relation exists, while in higher force ranges, no such relation could be established.

The role of reactive oxygen species in apoptosis of the diabetic kidney.

Increased levels of reactive oxygen species (ROS) by hyperglycemia can induce apoptosis of renal cells and diabetic nephropathy. The redox balance in the renal cell seems, therefore, of the utmost importance. ROS-mediated apoptosis may be further aggravated by an inadequate cytoprotective response against ROS. When there are insufficient cytoprotective and ROS scavenging molecules, ROS lead to considerable cellular damage and to a point of no return in apoptosis. Induction of cytoprotective proteins may prevent or attenuate apoptosis, renal cell injury, and finally diabetic nephropathy. Here, we discuss some mechanisms of apoptosis and several strategies that have been probed to ameliorate, or to prevent apoptosis in the diabetic kidney.

Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation.

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and that this could be inhibited by co-administration of antioxidants N-acetyl-l-cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25-microM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO-activity protects against curcumin-induced apoptosis. Interestingly, exposure to curcumin maximally induced HO-1 protein and HO-activity at 10-15 microM, whereas, at a concentration of >20-microM curcumin HO-1-expression and HO-activity was negligible. NAC-mediated inhibition of 25-microM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity. Moreover pre-induction of HO-1 using 5-microM curcumin protected fibroblasts against 25-microM curcumin-induced apoptosis. On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-microM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated. In conclusion, curcumin treatment in high doses (>25 microM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.

[Phosphoinositide 3-kinase (PI3-K) expression. Tumorigenesis of epithelial carcinoma of the mouth]. / Phosphoinositid-3-Kinase- (PI3-K) Expression. Tumorgenese von Plattenepithelkarzinomen der Mundhöhle.

Phosphoinositide 3-kinase (PI3-K) is a heterodimeric enzyme involved in the regulation of mitogenesis, apoptosis, cell adhesion, and motility. PI3-K was suggested as a protooncogene in human cancer. To determine the expression of PI3-K during cancerogenesis and tumor invasion of HNSCC, we investigated normal and dysplastic epithelium of the oral cavity, squamous cell carcinoma and lymph node metastasis by immunohistochemistry. The strongest immunoreactivity for p85alpha and p110alpha was found in invasive tumors and their metastases. Carcinomas in situ showed a focal positivity. Dysplasias and normal epithelium reacted predominantly negatively. The PI3-K inhibitor LY294002 inhibited proliferation and invasion of the HNSCC cell line CAL-27 and induced apoptosis in vitro. Our data suggest PI3-K as a marker of malignancy and tumor invasion. We suggest including PI3-K in the multistep carcinogenesis model of HNSCC. In addition, PI3-K is a potential target for pharmacological intervention.

Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase.

Various pathologic conditions, such as hemorrhage, hemolysis and cell injury, are characterized by the release of large amounts of heme. Recently, it was demonstrated that heme oxygenase (HO), the heme-degrading enzyme, and heme are able to modulate adhesion molecule expression in vitro. In the present study, the effects of heme and HO on inflammation in mice were analyzed by monitoring the biodistribution of radiolabeled liposomes and leukocytes in conjunction with immunohistochemistry. Small liposomes accumulate in inflamed tissues by diffusion because of locally enhanced vascular permeability, whereas leukocytes actively migrate into inflammatory areas through specific adhesive interactions with the endothelium and chemotaxis. Exposure to heme resulted in a dramatic increase in liposome accumulation in the pancreas, but also intestines, liver, and spleen exhibited significantly increased vascular permeability. Similarly, intravenously administered heme caused an enhanced influx of radiolabeled leukocytes into these organs. Immunohistochemical analysis showed differential up-regulation of the adhesion molecules ICAM-1, P-selectin, and fibronectin in liver and pancreas in heme-treated animals. Heme-induced adhesive properties were accompanied by a massive influx of granulocytes into these inflamed tissues, suggesting an important contribution to the pathogenesis of inflammatory processes. Moreover, inhibition of HO activity exacerbated heme-induced granulocyte infiltration. Here it is demonstrated for the first time that heme induces increased vascular permeability, adhesion molecule expression, and leukocyte recruitment in vivo, whereas HO antagonizes heme-induced inflammation possibly through the down-modulation of adhesion molecules.

Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression.

Heme oxygenase (HO), by catabolizing heme to bile pigments, down-regulates cellular hemoprotein, hemoglobin, and heme; the latter generates pro-oxidant products, including free radicals. Two HO isozymes, the products of distinct genes, have been described; HO-1 is the inducible isoform, whereas HO-2 is suggested to be constitutively expressed. We studied the inducing effect of several metal compounds (CoCl(2), stannic mesoporphyrin, and heme) on HO activity. Additionally, we studied HO-1 expression in experimental models of adhesion molecule expression produced by heme in endothelial cells, and the relationship of HO-1 expression to the induced adhesion molecules. Flow cytometry analysis showed that heme induces intracellular adhesion molecule 1 (ICAM-1) expression in a concentration (10-100 microM)- and time (1-24 h)-dependent fashion in human umbilical vein endothelial cells. Pretreatment with stannic mesoporphyrin, an inhibitor of HO activity, caused a 2-fold increase in heme-induced ICAM-1 expression. In contrast, HO induction by CoCl(2) decreased heme-induced ICAM-1 expression by 33%. To examine the contribution of HO-1 and HO-2 to endothelial HO activity, specific antisense oligonucleotides (ODNs) of each isoform were tested for their specificity to inhibit HO activity in cells exposed to heme. Endothelial cells exposed to heme elicited increased HO activity, which was prevented (70%) by HO-1 antisense ODNs. HO-2 antisense ODN inhibited heme-induced HO activity by 21%. Addition of HO-1 antisense ODNs prevented heme degradation and resulted in elevation of microsomal heme. Western blot analysis showed that HO-1 antisense ODNs selectively inhibited HO-1 protein and failed to inhibit HO-2 protein. Incubation of endothelial cells with HO-1 antisense enhanced heme-dependent increase of ICAM-1. In contrast, addition of HO-2 antisense to endothelial cells failed to increase adhesion molecules. The role of glutathione, an important antioxidant, was examined on heme-induced ICAM-1 expression. Endothelial cells pretreated with a glutathione precursor, N-acetylcysteine, or glutathione ester, showed a decrease in heme-induced ICAM-1 expression of 37 and 44%, respectively, suggesting that the mechanism of ICAM-1 induction by heme may be partly dependent on the levels of antioxidant. It is possible that amelioration of the heme-induced oxidative stress and expression of ICAM-I is due, in part, to the induction of HO-1 activity. Regulation of HO activity in this manner may have clinical applications.

Distinct effect of retroviral-mediated IFN-alpha gene transfer on human erythroleukemic and CD34+ cell growth and differentiation.

Human interferon-alpha (IFN-alpha) has been used in the management of leukemia, but its diverse adverse effects may influence the ability of IFN-alpha to treat this disease. We constructed two retroviral vectors, LSN-IFN-alpha and LNC-IFN-alpha, in which IFN-alpha cDNA was driven by viral LTR and CMV promoters, respectively. After transduction into the PA317 and PG13 retroviral packaging cells, high titers of retrovirus were produced and were used to infect K562 and human BM CD34+ hematopoietic cells. The IFN-alpha gene expression in transduced K562 cells was confirmed by Northern blot, RT-PCR, RIA, and biologic assay. Cell proliferation and cell viability in IFN-alpha-transduced K562 cells were significantly suppressed as compared with control K562 cells. Although the IFN-alpha expression in K562 cells did not affect BCR/ABL expression, it apparently upregulated the production of adhesion molecules (VLA-4 and Mac-1). We evaluated the effect of IFN-alpha gene transfer on human CD34+ cells infected with LSN-IFN-alpha retrovirus with the aid of fibronectin (FN) fragment CH-296 and growth factors. RIA showed that IFN-alpha-transduced CD34+ cells produced 72.2+/-15 U/ml of IFN-alpha compared with 4.3+/-1.2 U/ml in control CD34+ cells. Methylcellulose clonogenic assay indicated that IFN-alpha-transduced CD34+ cells produced similar numbers of burst-forming units-erythrocytes (BFU-E)/colony-forming units-GM (CFU-GM) colonies as compared with control CD34+ cells. Selected colonies expressed IFN-alpha and neo(r) mRNA, as measured by RT-PCR. These studies indicate that retrovirus-mediated IFN-alpha gene transfer may provide a useful tool for studying the effect of IFN-alpha gene transfer on leukemic cells and long-lived CD34+ cells.

The Epstein-Barr virus BARF1 gene encodes a novel, soluble colony-stimulating factor-1 receptor.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with infectious mononucleosis and several tumors. The BARF1 gene is transcribed early after EBV infection from the BamHI A fragment of the EBV genome. Evidence shown here indicates that the BARF1 protein is secreted into the medium of transfected cells and from EBV-carrying B cells induced to allow lytic replication of the virus. Expression cloning identified colony-stimulating factor-1 (CSF-1) as a ligand for BARF1. Computer-assisted analyses indicated that subtle amino acid sequence homology exists between BARF1 and c-fins, the cellular proto-oncogene that is the receptor for CSF-1. Recombinant BARF1 protein was found to be biologically active, and it neutralized the proliferative effects of human CSF-1 in a dose-dependent fashion when assayed in vitro. Since CSF-1 is a pleiotropic cytokine best known for its differentiating effects on macrophages, these data suggest that BARF1 may function to modulate the host immune response to EBV infection.

Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells.

Heme is an important immunostimulating agent and oxidative factor contributing to endothelial cell activation. To investigate the mechanism of heme-induced endothelial cell activation, we analyzed the effect of heme and the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), on the expression of the heme-degrading stress protein, heme oxygenase (HO), and adhesion molecules in human umbilical vein endothelial cells (HUVEC). Indirect immunofluorescence double labeling studies demonstrated a simultaneous increase of ICAM-1 and HO-1 after exposure of cells to heme for 24 hr. Co-expression of HO-1 and ICAM-1 was also demonstrated in TNF-alpha-exposed cells. Dot blot immunoassay and quantitative analysis by ELISA demonstrated that heme treatment for 24 hr caused a 2-fold increase in ICAM-1 expression (P < 0.002) compared with quiescent cells, while in cells stimulated by TNF-alpha for 24 hr ICAM-1 gene expression increased by 5-fold. Moreover, heme exposure also resulted in a marked increase in VCAM-1 and E selectin expression (three and four times over control levels, respectively). On the other hand, TNF-alpha treatment showed similar expression levels for VCAM-1 and E selectin, compared with stimulation by heme (100 microM). The level of HO activity in endothelial cells exposed to heme or TNF-alpha was increased from 24.7 +/- 5.7 pmol bilirubin/mg protein/min in control to 70.0 +/- 9.5 and 36.7 +/- 3.1 pmol bilirubin/mg protein/min in heme- and TNF-alpha-stimulated cells, respectively. These results suggest that upregulation of ICAM-1, VCAM-1, and E selectin expression is associated with oxidative stress induced by hemoglobin/heme and that HO-1 may play a modulating role via its ability to degrade heme to a substance with antioxidant properties.