Selective induction of nerve growth factor and brain-derived neurotrophic factor by LPS and allergen in dendritic cells.

BACKGROUND: Neurotrophins are produced by various cells upon different stimuli and participate in the initiation and regulation of inflammation in various diseases including allergy and asthma, but little is known about the production and control of neurotrophins by dendritic cells (DCs). The aim of this study was to assess whether DCs produce the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and whether inflammatory stimuli or allergens are able to induce the production of neurotrophic factors. METHODS: Monocyte-derived dendritic cells (MoDCs) were generated from different donors. The neurotrophins NGF and BDNF were demonstrated by RT-PCR, Western blotting, flow cytometry analysis and fluorescence microscopy. MoDCs were cultured and stimulated with lipopolysaccharide (LPS) or allergen for 24 h. The supernatants and cells were collected. Measurement for NGF and BDNF was performed by ELISA. RESULTS: DCs express mRNA for the neurotrophins NGF and BDNF. Proteins were detectable by Western blot, FACS analysis and fluorescence microscopy. LPS led to an up-regulation of BDNF, while NGF was unaffected. Cell lysates demonstrated an increased amount of BDNF after stimulation with LPS or allergen, while NGF was not affected significantly. CONCLUSIONS: DCs are a source of neurotrophins. LPS selectively regulates the production of BDNF. Allergen stimulation leads to an LPS-independent regulation. This contributes to a complex involvement of neurotrophins in allergic diseases.

A skeletal muscle-specific enhancer regulated by factors binding to E and CArG boxes is present in the promoter of the mouse myosin light-chain 1A gene.

The mouse myosin light-chain 1A (MLC1A) gene, expressed in the atria of the adult heart, is one of the first muscle genes to be activated when skeletal as well as cardiac muscles form in the embryo. It is also transcribed in skeletal muscle cell lines at the onset of differentiation. Transient transfection assays of mouse skeletal muscle cell lines with DNA constructs containing MLC1A promoter fragments fused to the chloramphenicol acetyltransferase (CAT) gene show that the first 630 bp of the promoter is sufficient to direct expression of the reporter gene during myotube formation. Two E boxes located at bp -76 and -519 are necessary for this regulation. MyoD and myogenin proteins bind to them as heterodimers with E12 protein and, moreover, transactivate them in cotransfection experiments with the MLC1A promoter in nonmuscle cells. Interestingly, the effect of mutating each E box is less striking in primary cultures than in the C2 or Sol8 muscle cell line. A DNA fragment from bp -36 to -597 confers tissue- and stage-specific activity to the herpes simplex virus thymidine kinase promoter in both orientations, showing that the skeletal muscle-specific regulation of the MLC1A gene is under the control of a muscle-specific enhancer which extends into the proximal promoter region. At bp -89 is a diverged CArG box, CC(A/T)6AG, which binds the serum response factor (SRF) in myotube nuclear extracts, as does the wild-type sequence, CC(A/T)6GG. Both types of CArG box also bind a novel myotube-enriched complex which has contact points with the AT-rich part of the CArG box and adjacent 3' nucleotides. Mutations within the CArG box distinguish between the binding of this complex and binding of SRF; only SRF binding is directly involved in the specific regulation of the MLC1A gene in skeletal muscle cell lines.

Cyclic GMP-activated protein kinase from Dictyostelium discoideum.

Cells of Dictyostelium discoideum respond to their chemoattractants, cAMP and folate, with a rapid increase of the cellular cGMP content. The molecular mechanisms of cGMP action are not understood. Since in many biological systems cGMP-activated protein kinase is a prominent cGMP acceptor, we searched for such an enzyme in D. discoideum. By means of affinity chromatography on cGMP-Sepharose and other chromatographic procedures (DEAE-Trisacryl, CM-Trisacryl), we separated a novel protein kinase. This preparation did not show any regulation by cGMP and may represent an enzyme modified by proteolysis. In order to establish a rapid and efficient purification step, an antiserum against the kinase preparation was raised and coupled to Sepharose. Chromatography of the supernatant from a cell homogenate on this antibody matrix yielded a protein kinase that was activated 3-fold by cGMP. Half-maximal activation occurred at about 1 nM cGMP. Cyclic AMP at a 20-fold higher concentration also activated the protein kinase. On a Superose 6HR column the cGMP-activated protein kinase eluted in the same volume as enolase (Mr = 82,000).

Retinoic acid affects the expression rate of the differentiation-related genes aryl hydrocarbon receptor, ARNT and keratin 4 in proliferative keratinocytes only.

The environmental contaminant dioxin exerts most of its effects by activating the aryl hydrocarbon receptor (AhR). The AhR is considered to play not only a role in the regulation of xenobiotic metabolism, but also for development, growth, and differentiation. The transcript levels of the AhR and its associated translocator protein (ARNT) were found to increase with ongoing differentiation in the human keratinocyte cell line HaCaT. Correspondingly, in situ hybridization studies in normal human skin revealed an absence of AhR-expression in proliferating basal cells and increasing transcript levels in upper cell layers, in dependence of keratinocyte differentiation. AhR expression in differentiation-deficient hyperproliferative psoriatic skin was markedly decreased. When keratinocytes were continuously treated with 1 microM retinoic acid (RA), the upregulation of AhR- and ARNT-mRNA levels was inhibited as was keratin 4-expression, a marker of HaCaT-keratinocyte differentiation. In contrast, treatment of already differentiated cells with RA did not down-regulate these transcript levels. The mRNA levels of the prevalent retinoic acid receptors in keratinocytes, RAR gamma and RXR alpha, were not influenced by the process of differentiation or by addition of RA. Our data suggest that the regulation of AhR-, ARNT- and keratin 4-expression by RA is indirect and mediated by a yet to be identified factor.

Proliferation-dependent induction of apoptosis by the retinoid CD437 in p53-mutated keratinocytes.

6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-Naphthalene carboxylic acid (CD437) is a synthetic retinoid with strong apoptogenic properties in various neoplastic cell lines. CD437 was shown to induce apoptosis in malignant human keratinocytes but not in normal keratinocytes. We demonstrate that CD437 is also capable of inducing apoptosis in the non-tumorigenic keratinocyte cell line HaCaT that carries UV-type mutations on both alleles of the p53 gene. The concentration-dependent induction of apoptosis was restricted to proliferative HaCaT cells, whereas no effect was seen in differentiating post-mitotic cells. The apoptotic elimination of the proliferative cells was accompanied by rapid upregulation of c- jun, downregulation of c- fos, and activation of the AP-1 complex, which normally only occur during the differentiation process of post-mitotic keratinocytes. Pharmacological impairment of this precocious AP-1 activation reduced the rate of apoptosis induced by CD437. The potent, selective, and p53-independent apoptosis-inducing efficacy of CD437 is of utmost importance for the prophylaxis and treatment of skin cancer caused by mutational inactivation of the p53 gene.

Is CT cystography an accurate study in the evaluation of spontaneous perforation of augmented bladder in children and adolescents?

BACKGROUND: Spontaneous bladder perforation (SBP) is a potentially fatal complication of augmented bladder. Imaging is often used for diagnosis. In this study we present our experience with CT cystography (CTC) in the diagnosis of SBP. OBJECTIVE: To determine CTC accuracy in the evaluation of SBP in children with an augmented bladder. STUDY DESIGN: The institutional review board approved this HIPAA-compliant study; informed consent was waived. All patients under 20 years old, who underwent CTC for SBP evaluation from 2003 to 2013, were identified. Two radiologists independently reviewed CTC studies for contrast extravasation, ascites, and pneumoperitoneum. Ascites was graded: small - confined to the rectovesical pouch (RVP); moderate - beyond the RVP; large - beyond the pelvis. RESULTS: Eighty-nine patients (47 males, age 4.2-19.8 years) had 132 CTCs. SBP was diagnosed in 14% (19/132). Both radiologists found contrast extravasation in 74% (14/19) of patients with SBP; two patients had only pneumoperitoneum, and three had only ascites (large = 2, moderate = 1) (Fig.). SBP was found in 1% of CTCs with no ascites or small ascites (1 of 98 and 92; radiologists 1 and 2, respectively). Findings of extraluminal extravasation, unexplained pneumoperitoneum, or large ascites, yielded a detection rate of 95% for SBP by each radiologist. In eight patients, small bowel obstruction was diagnosed. DISCUSSION: Contrast extravasation was detected in only 74% of patients with SBP. The use of indirect signs of perforation (unexplained pneumoperitoneum and large ascites) in addition to contrast extravasation, increased the detection rate of SBP to 95%. US screening for SBP and selection of patients with moderate or large ascites for CTC, may eliminate the need for most CT scans. In the absence of SBP, other abdominal abnormalities should be evaluated. Bowel obstruction was the most common non-urological emergency detected in this series. The main limitations of the study are: the small number of SBP cases; the diagnosis of SBP not based on surgical findings in three patients; and inability to completely exclude occult SBP in patients not explored surgically. CONCLUSION: Extraluminal contrast was seen on CTC in most cases of SBP, but some patients with sealed bladder perforation had only pneumoperitoneum or moderate/large ascites. Therefore, SBP should be suspected in any patient with moderate/large volumes of pelvic fluid or unexplained pneumoperitoneum, even when there is no evidence of contrast extravasation. Patients with no ascites, or small volumes, are unlikely to have SBP; therefore, US can be used to screen low risk patients.

Allelic variations of human keratins K4 and K5 provide polymorphic markers within the type II keratin gene cluster on chromosome 12.

To appreciate point mutations in keratin genes as causes for hereditary epithelial diseases, the normal variation of these gene sequences in the population must be known. Because genetic polymorphism of keratins at the protein level due to allelic variation has been described for the type II keratins 4 and 5, we have analyzed their corresponding genes using single-strand conformation polymorphism gel electrophoresis and sequence analysis of polymerase chain reaction amplified genomic DNA. Although no sequence variations were found in the carboxyl-terminal and rod domains we were able to map the molecular differences among the alleles to their amino-terminal domains. In particular, we have identified three alleles of keratin 4. Two alleles differed by a nucleotide transition causing a neutral amino acid substitution (alanine to valine) and one allele had a 42-bp in-frame deletion corresponding to 14 amino acids within the V1 subdomain. Three alleles were also recognized for the keratin 5 locus, all being elicited by single nucleotide substitutions. Of these, only one altered the amino acid sequence, replacing an uncharged (glycine) with a charged (glutamic acid) amino acid in the H1 subdomain. Pedigree analyses in three families showed the alleles to be inherited as autosomal Mendelian traits. Thus, these normal alleles of keratins 4 and 5 will provide favorable polymorphic markers for linkage analysis directly within the cluster of type II keratin genes located on chromosome 12q to elucidate the potential involvement of these and other keratin genes in disorders of squamous cell differentiation.

Role of p53 in UVB-induced apoptosis in human HaCaT keratinocytes.

Apoptosis represents an active form of cell death that is involved in the control of tissue homeostasis and in the deletion of DNA-damaged cells. Because the product of the tumor suppressor gene p53 has been demonstrated to be crucial for the induction of apoptosis in certain cell types, the present study was aimed at elucidating its role in ultraviolet-induced apoptosis in HaCaT keratinocytes. After in vitro ultraviolet B irradiation, p53 protein levels were noted to increase prior to the induction of apoptosis in a time- and concentration-dependent fashion. This increase could not be inhibited by the protein synthesis inhibitor cycloheximide. Because HaCaT keratinocytes are known to bear two p53 point mutations and because it is unclear whether p53 in HaCaT cells is still functional regarding induction of apoptosis, HaCaT cells were stably transfected with wild-type p53 cDNA inserted into the expression vector pCMV-Neo-Bam in sense (pC53-SN3) and anti-sense (pC53-ASN) direction. After selection with geniticin, growing colonies were screened for the presence of the transfected cDNA constructs by polymerase chain reaction. Cell clones bearing the anti-sense product were further analyzed for p53 expression by western blotting. Clones showing reduced p53 protein levels were irradiated with ultraviolet B light, and there was a clear reduction of apoptosis in the pC53-ASN bearing cell clones compared with the parental HaCaT cells. These studies demonstrate that blocking mutated p53 can partially block apoptosis in HaCaT keratinocytes and furthermore can confirm the key role for p53 in ultraviolet-induced apoptosis in human keratinocytes. Moreover, HaCaT keratinocytes and their p53-transfectants provide a convenient model that allows for further detailed analyses of apoptosis-associated biochemical and molecular events in human keratinocytes.

Keratin 17 gene expression during the murine hair cycle.

Keratin 17 (K17) expression is currently considered to be associated with hyperplastic or malignant growth of epithelial cells. The functions of this keratin in normal skin physiology and the regulation of its gene expression, however, are still unclear. As one possible approach to further explore K17 functions, we have studied the differential patterns of mouse K17 (MK17) transcription during the murine hair cycle by means of in situ hybridization, using a digoxigenin-labeled riboprobe. Cycling hair follicles in the skin of C57BL/6 mice were found to be the only skin structures expressing MK17 under physiologic conditions. MK17 transcripts were constantly observed throughout all hair cycle stages in the suprainfundibular outer root sheath (ORS). The MK17 expression was also evident in the isthmus part of the ORS, where it was expressed weakly and was spatially restricted during telogen, with an increase in early anagen and stable expression during mid- and late anagen, localizing to the zone of so-called trichilemmal keratinization. In addition, in early anagen, a group of epithelial cells in or next to the bulge region stained weakly for MK17. With progressing anagen development, MK17 expression in this region increased and was consistently localized to keratinocytes at the advancing front of the emerging epithelial hair bulb. In mid- and late anagen, this zone of MK17 expression spread along the proximal ORS, with a maximal level of expression in the innermost cell layer of the ORS. Overall, these findings provide data on the MK17 expression profile of normal murine skin and demonstrate hair-cycle-dependent regulation of MK17 expression.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCCD) affects keratin 1 and keratin 17 gene expression and differentially induces keratinization in hairless mouse skin.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes chloracne in humans by mechanisms that are as yet poorly understood. Because TCDD is known to affect keratinocyte differentiation in vitro, we have studied TCDD-dependent morphologic changes and the expression of murine keratin 1 (MK1; differentiation associated) and keratin 17 (MK17; presumably hyperproliferation associated) in HRS/J hr/hr hairless mouse skin. TCDD (0.2 microg in acetone) applied topically to the dorsal skin caused epidermal acanthosis and hyperkeratosis of the dermal cysts as well as an involution of the utricles and the sebaceous glands. By means of in situ hybridization with digoxigenin-labeled riboprobes of sections from untreated and vehicle (control)-treated skin, we localized MK1 mRNA to the epidermal spinous cell compartment. MK17 transcripts were detected only in the derivatives of the hair follicle-utricle epithelium and dermal cysts. No spatial overlap was observed between MK1 and MK17 expression. After TCDD application, MK17 was newly expressed in the upper spinous cell layers of the interfollicular epidermis, although it was suppressed in the involuting utricles. In contrast, MK1 expression in the interfollicular epidermis was not affected by TCDD. Furthermore, MK1 expression was induced in the epithelium of the utricle remnants and in some dermal cysts. These data suggest that increased keratinization of the part of the follicular epithelium corresponding to the dermal cyst epithelium of hairless mice most probably explains the pathogenesis of TCDD-induced chloracne. The results demonstrate, furthermore, that TCDD can differentially affect keratinocyte differentiation in vivo as well as in vitro.

The loss of desmosomes after retinoic acid treatment results in an apparent inhibition of HaCaT keratinocyte differentiation.

Epithelial tissue cohesion is based on various types of intercellular adhering junctions of which the desmosomes are particularly abundant in stratified epithelia. The desmogleins (dsg) and desmocollins are their transmembrane components. One or more of the three isoforms of these desmosomal cadherins are co-expressed and specific subtypes prevail at different stages of epidermal differentiation. In HaCaT keratinocytes, desmosomal cadherin expression increased with ongoing differentiation, apart from dsg2. Continuous treatment with retinoic acid (RA) inhibits the differentiation of HaCaT keratinocyte cultures. RA strongly increased the shedding of cells into the culture medium where they quickly underwent cellular death. Electron microscopy showed a marked reduction of desmosomes with nearly complete absence of their structural components, suggesting that RA inhibits their synthesis. RA indeed downregulated the transcript levels of all HaCaT desmosomal cadherins, except dsg2. Immunostaining revealed that desmosomal protein contents corresponded to alterations in transcription rates. Our findings indicate that the RA-induced inhibition of differentiation of keratinocyte cultures results from removal of cells committed to differentiation. In vivo, less adhering but still differentiating cells cannot be removed as easily as they can be in a culture system. The consequence is a sticky and fragile skin.

The promoter of the HaCaT keratinocyte differentiation-related gene keratin 4 contains a functional AP-2 binding site.

The nuclear transcription factor AP-2 appears to be a key regulator mediating programmed gene expression during embryonic morphogenesis and adult cell differentiation. AP-2 has also been considered to be involved in epidermal gene regulation, but its precise role is not yet defined. The level of AP-2 transcripts increases during culturing of HaCaT keratinocytes preceding the expression of the differentiation-related gene keratin 4 (K4). The current study was aimed at investigating whether AP-2 transactivates K4 transcription. We cloned and sequenced the promoter region of K4 and found, in addition to canonical sequences, an AP-2 consensus site in the vicinity of the transcriptional start. In order to provide functional evidence for a regulation of K4 transcription by AP-2, we cloned various parts, which did or did not contain the AP-2 site of the K4 upstream sequence, into Cat reporter plasmids. These constructs were ballistically transfected into differentiating HaCaT keratinocytes. The determination of the resulting Cat activity revealed that the AP-2 site in the vicinity of the transcriptional start was functional for K4 transcription. Thus, the role of AP-2 in the process of keratinocyte differentiation appears to be considerable. In addition, further regulatory elements were found to be necessary for full transcription of K4.

Comparison of the effects of radiation and hyperthermia on prenatal retardation of brain growth of guinea-pigs.

On day 21 of pregnancy guinea-pigs were exposed to hyperthermia or gamma radiation. The effects on prenatal growth and especially brain growth of offspring were compared. Doses of 0.04-0.99 Gy of radiation produced a dose-dependent and irreversible reduction of brain weight in the offspring, but had little effect on body weight. Treatment with hyperthermia resulting in maternal temperatures of 41.8-43.9 degrees C after exposure in a heated incubator for an hour also produced a dose-related micrencephaly in the offspring. Comparison of the two agents showed that a dose increment of 0.525 Gy of radiation produced a deficit in brain weight equivalent to an elevation of 1 degree C in maternal temperature. Using this guinea-pig brain weight assay system a threshold was detected of between 0.05 and 0.10 Gy for retardation of brain growth.

Social and economic context and attitudes toward immigrants in Canadian cities.

PIP: This article presents a study of the effects of a number of socioeconomic features of Canadian cities, particularly their unemployment rates, on the attitudes toward immigrants of their native-born residents. Using data from a national study of ethnicity and multiculturalism, the authors estimate several regression models predicting 3 separate dimensions of attitude toward immigrants and including as independent variables both individual characteristics and structural characteristics of city of residence. They found no evidence of a sizeable effect of local unemployment rate on attitude toward immigrants. Of the other contextual variables included in the models, the only 2 consistently influencing these attitudes included in the models, educational attainment and income, along with mother tongue, exhibit the strongest and most consistent effects on the attitude dimensions.^ieng