Influence of the skin barrier on the penetration of topically-applied dexamethasone probed by soft X-ray spectromicroscopy.

The penetration of dexamethasone into human skin ex vivo is reported. X-ray microscopy is used for label-free probing of the drug and quantification of the local drug concentration with a spatial resolution reaching 70±5nm. This is accomplished by selective probing the dexamethasone by X-ray absorption. Varying the penetration time between 10min and 1000min provides detailed information on the penetration process. In addition, the stratum corneum has been damaged by tape-stripping in order to determine the importance of this barrier regarding temporally resolved drug penetration profiles. Dexamethasone concentrations distinctly vary, especially close to the border of the stratum corneum and the viable epidermis, where a local minimum in drug concentration is observed. Furthermore, near the basal membrane the drug concentration strongly drops. High spatial resolution studies along with a de-convolution procedure reveal the spatial distribution of dexamethasone in the interspaces between the corneocytes consisting of stratum corneum lipids. These results on local drug concentrations are interpreted in terms of barriers affecting the drug penetration in human skin.

State of the art in neuroprotection during acute type A aortic dissection repair.

Temporary (TND) or permanent neurologic dysfunctions (PND) represent the main neurological complications following acute aortic dissection repair. The aim of our experimental and clinical research was the improvement and update of the most common neuroprotective strategies which are in present use. HYPOTHERMIC CIRCULATORY ARREST (HCA): Cerebral metabolic suppression at the clinically most used temperatures (18-22°C) is less complete than had been assumed previously. If used as a 'stand-alone' neuroprotective strategy, cooling to 15-20°C with a jugular SO(2) ≥ 95% is needed to provide sufficient metabolic suppression. Regardless of the depth of cooling, the HCA interval should not exceed 25 min. After 40 min of HCA, the incidence of TND and PND increases, after 60 min, the mortality rate increases. ANTEGRADE SELECTIVE CEREBRAL PERFUSION (ASCP): At moderate hypothermia (25-28°C), ASCP should be performed at a pump flow rate of 10ml/kg/min, targeting a cerebral perfusion pressure of 50-60mmHg. Experimental data revealed that these conditions offer an optimal regional blood flow in the cortex (80±27ml/min/100g), the cerebellum (77±32ml/min/100g), the pons (89±5ml/min/100g) and the hippocampus (55±16ml/min/100g) for 25 minutes. If prolonged, does ASCP at 32°C provide the same neuroprotective effect? CANNULATION STRATEGY: Direct axillary artery cannulation ensures the advantage of performing both systemic cooling and ASCP through the same cannula, preventing additional manipulation with the attendant embolic risk. An additional cannulation of the left carotid artery ensures a bi-hemispheric perfusion, with a neurologic outcome of only 6% TND and 1% PND. NEUROMONITORING: Near-infrared spectroscopy and evoked potentials may prove the effectiveness of the neuroprotective strategy used, especially if the trend goes to less radical cooling. CONCLUSION: A short interval of HCA (5 min) followed by a more extended period of ASCP (25 min) at moderate hypothermia (28°C), with a pump flow rate of 10ml/kg/min and a cerebral perfusion pressure of 50 mmHg, represents safe conditions for open arch surgery.

Goal-directed hemodynamic optimization in high-risk cardiac surgery patients: a tale from the past or a future obligation?

The present manuscript summarizes the available evidence on outcome-related hemodynamic variables and ''goal-directed hemodynamic optimization'' strategies in patients undergoing cardiac surgery.

Preexisting atrial fibrillation as predictor for late-time mortality in patients with end-stage renal disease undergoing cardiac surgery--a multicenter study.

BACKGROUND: Although patients with end-stage renal disease (ESRD) are considered to be high-risk patients in cardiac surgery, the reported studies are rather small, resulting in unsatisfactory analyses of outcome determinants. Therefore, we aimed to identify possible risk factors, with a particular focus on the impact of pre-existing atrial fibrillation (AF) on the postoperative short-term and long-term mortality of ESRD patients undergoing cardiac surgery. METHODS: In a multicenter study 522 patients with ESRD undergoing CABG only (62.9 %), valve surgery only (17.2 %), or both (19.9 %) with comparable demographic and other cardiac risk factor characteristics were investigated retrospectively over a period of 10 years. The outcome was divided into perioperative (within 30 days) and late morbidity and mortality, and multivariate analysis was performed for both. RESULTS: The mean perioperative mortality was 11.5 % and the 5-year survival rate was 42 %. Emergency surgery, insulin-dependent diabetes mellitus, the number of vein grafts and age were identified as risk factors whereas complete revascularization, the use of an internal thoracic artery and the presence of sinus rhythm were identified as beneficial factors for long-term survival. 14.1 % of all patients had pre-existing AF. Although AF was not identified as an independent risk factor for perioperative mortality ( P = 0.59), it was identified as an independent predictor for late mortality ( P < 0.001). Median survival of patients without AF was 1816 days, while for patients with AF it was only 715 days. CONCLUSIONS: AF does represent an independent predictor for long-term but not perioperative mortality in patients with ESRD. However, effective treatment of AF is controversially discussed. Anticoagulation therapy or perioperative ablation of the arrhythmia should be considered in order to improve the survival of these patients.

Types of reconstructive surgery of the aortic valve.

The aortic valve is part of the aortic root which is wedged between the heart and the ascending aorta, maintaining a directional flow throughout life-span. Beside different types of aortic valve replacements, reconstructive techniques are increasingly performed to restore normal aortic valve function. To apply these operations, understanding of normal and pathological valve anatomy and physiology is of basic importance. In addition, a widely accepted uniform aortic valve and root terminology is desirable for a proper scientific communication. Reconstructive techniques themselves can be divided into isolated reconstruction of aortic valve/root structures and the isolated replacement of one or more structures. Examples for the former ones are commissurotomy, cusps plication, decalcification or extension as well as plications of other aortic root structures (i.e. the intercusp triangles or the basal annulus). Examples for the latter ones are the remodeling and reimplantation techniques and their modifications. Replacement of the ascending aorta at the sinotubular level for the adjustment of the commissures to restore aortic root geometry also belongs to this group of techniques for aortic valve reconstruction. In this review article a systematic description of the current reconstructive techniques to restore adequate aortic valve function as well as clinical data are presented.

Serum S100B levels in patients after cardiac surgery: possible sources of contamination.

BACKGROUND: S100B protein is considered to be a potential marker of brain damage. The aim of our study was to determine the contamination effect of retransfused blood on the serum S100B concentrations in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and to differentiate between this simple contamination effect and its possible enhancement by haemolysis. METHODS: The first part of the study was performed in a group of 10 patients scheduled for coronary artery bypass grafting. Baseline S100B level was determined in a blood sample drawn from the radial artery before skin incision. After performing the distal anastomosis, additional blood samples were drawn from 1) the radial artery, 2) the aortic root catheter, 3) the pericardial space, and 4) CPB suction. To study the possible haemolytic effect on serum S100B levels, a second group of 23 patients was studied. S100B concentrations were determined in samples drawn simultaneously from the radial artery and bypass circuit after the end of CPB. Further samples from the retransfusion blood bag were analysed after one, two and three hours. RESULTS: Blood samples from the pericardial space and CPB suction exhibited significantly higher levels of S100B than the samples drawn from the peripheral artery and aortic root catheter in the first group of patients. No significant differences between the S100B levels in the peripheral blood and aortic root catheter were detected. In the second group, S100B was significantly elevated in the samples taken from the retransfusion blood bag in comparison with peripheral blood. S100B levels remained stable during the whole follow-up period. CONCLUSION: The results of our study show increased serum S100B levels caused by contamination originating in the mediastinal tissues. Storage of blood in the retransfusion bag and haemolysis can be excluded as sources of contamination. The role of S100B in perioperative monitoring of patients undergoing cardiac surgery remains to be established and should be confirmed by further studies using neuropsychological tests and imaging techniques.

CUTL1 promotes tumor cell migration by decreasing proteasome-mediated Src degradation.

Recently, we identified the homeodomain transcription factor CUTL1 as important mediator of cell migration and tumor invasion downstream of transforming growth factor beta (TGFbeta). The molecular mechanisms and effectors mediating the pro-migratory and pro-invasive phenotype induced by CUTL1 have not been elucidated so far. Therefore, the aim of this study was to identify signaling pathways downstream of CUTL1 which are responsible for its effects on tumor cell migration. We found that the reduced motility seen after knock down of CUTL1 by RNA interference is accompanied by a delay in tumor cell spreading. This spreading defect is paralleled by a marked reduction of Src protein levels. We show that CUTL1 leads to Src protein stabilization and activation of Src-regulated downstream signaling molecules such as RhoA, Rac1, Cdc42 and ROCK. In addition, we demonstrate that CUTL1 decreases proteasome-mediated Src protein degradation, possibly via transcriptionally upregulating C-terminal Src kinase (Csk). Based on experiments using Src knockout cells (SYF), we present evidence that Src plays a crucial role in CUTL1-induced tumor cell migration. In conclusion, our findings linking the pro-invasive transcription factor CUTL1 and the Src pathway provide important new insights in the molecular effector pathways mediating CUTL-induced migration and invasion.

Decellularized pulmonary homograft (SynerGraft) for reconstruction of the right ventricular outflow tract: first clinical experience.

INTRODUCTION: Cryopreserved homograft valve conduits have been used to reconstruct the right and left ventricular outflow tract. Long-term studies have shown homograft degeneration and calcification, and it has been postulated that immunological mediated phenomena in a manner similar to that seen in chronic rejection may contribute to the degeneration process. The development of a decellularized, non-glutaraldehyde-fixed valve conduit creates a non-immunogenic connective tissue matrix for autologous recellularization by host cells. The aim of the study was to characterize the clinical and hemodynamic pattern in human implants of the novel decellularized pulmonary homografts (SynerGraft). METHODS: Reconstruction of the right ventricular outflow tract was performed in 17 patients: 15 patients with aortic valve disease and the Ross procedure, and two patients with redo procedures following Fallot tetralogy and severe pulmonary regurgitation. Patients with the Ross procedure with standard cryopreserved homografts as neopulmonic conduits served as controls. Within the follow-up over six months morphological and hemodynamic parameters were characterized by echocardiography: maximal and mean pressure gradient across the right and left ventricular outflow tract, their effective orifice areas, determination of neopulmonic and neoaortic regurgitation. RESULTS: One patient died six weeks following surgical treatment due to non-valve related end-stage cardiopulmonary failure; all patients were free of valve-related complications during the follow-up period. The matched Ross patients showed a gradual but significant increase of both the maximal and mean pressure gradient across the right ventricular outflow tract (Delta P max 5.5+/-2.5 to 11.4+/-6.4 mmHg, p=0.002; Delta P mean 3.0+/-1.3 to 6.2+/-3.9 mmHg, p=0.003), whereas in the SynerGraft group increase of pressure gradients were measurable but did not reach statistical significance (Delta P max 7.1+/-3.7 to 10.1+/-3.9 mmHg, p=0.11; Delta P mean 3.6+/-1.6 to 5.5+/-2.3 mmHg, p=0.12). The pulmonary effective orifice areas decreased in the control group from 1.74+/-0.33 to 1.18+/-0.36 cm(2)/m(2) (p=0.001). Within the SynerGraft group time dependent reduction of the orifice area was significantly less (1.51+/-0.37 to 1.25+/-0.26 cm(2)/m(2); p=0.08). CONCLUSION: Up to six months after implantation reconstruction of the right ventricular outflow tract with decellularized homografts was safe, stable, and the morphological and hemodynamic features are promising.

Time course of aortic valve function and root dimensions after subcoronary ross procedure for bicuspid versus tricuspid aortic valve disease.

BACKGROUND: The freestanding aortic root, which is the currently preferred operative technique for pulmonary autografts, is reported to dilate and potentially promote aortic insufficiency, which has led to a controversial debate on the appropriate surgical technique, especially for congenital bicuspid aortic valve disease. Desirable data on the time course of valve function and root dimensions for the alternative subcoronary technique comparing bicuspid and tricuspid aortic valve disease are scarce. METHODS AND RESULTS: Echocardiographic examinations of 31 patients with congenital bicuspid aortic valve disease (group A; age 50.5+/-11.0 years) and 51 patients with acquired tricuspid aortic valve disease (group B; age 48.1+/-15.7 years) who were operated on between June 1994 and August 1998 were performed twice postoperatively. At first and second follow-up, respectively, maximum (mean) pressure gradients were 6.0+/-2.0 (3.6+/-1.0) and 5.1+/-2.1 (2.9+/-1.1) mm Hg in group A and 6.5+/-3.5 (3.9+/-1.9) and 5.0+/-1.7 (2.9+/-1.0) mm Hg in group B (P>0.05 between groups). In group A, grade 0 aortic insufficiency at first and second follow-up occurred in 8 and 7 patients, respectively, grade 0-I in 12 and 9 patients, grade I in 9 and 11 patients, grade I-II in 1 and 0 patients, and grade II in 1 and 4 patients; in group B, grade 0 aortic insufficiency occurred in 16 and 18 patients, grade 0-I in 16 and 8 patients, grade I in 17 and 21 patients, grade I-II in 0 and 1 patient, and grade II in 0 and 1 patient (P>0.05). Aortic insufficiency decreased in 10 patients (17%). However, there was an overall tendency for aortic insufficiency to increase over time (n=23, 38%), although it remained subclinical. Aortic root dimensions did not differ between groups and were constant during follow-up. CONCLUSIONS: This study provides some evidence that the function of the subcoronary pulmonary autograft in bicuspid aortic valve disease is excellent, with stable root dimensions, and is not different from that of tricuspid aortic valves at least up to 5.5 years postoperatively, which suggests the subcoronary technique should be reconsidered.

Molecular and functional characterization of the four-transmembrane molecule l6 in epidermal keratinocytes.

In normal human epidermal keratinocytes (NHEK) proteolytic detachment from the substrate induces a complex activation cascade including expression of new proteins, morphological alterations, and the onset of migration for epidermal regeneration. By subtractive cloning we have shown that L6, a four-transmembrane protein, is newly expressed after proteolytic keratinocyte detachment. In this study, we have generated a novel anti-L6 antibody (clone HD-pKe#104-1.1) and investigated L6 expression regulation in vitro and in vivo as well as L6 function in keratinocyte migration. Dispase-mediated detachment induced L6 expression in NHEK at the mRNA and protein level. Immunohistology of skin biopsies displayed a strong expression of L6 in follicular epidermis and epidermolytic lesions of autoimmune bullous dermatoses (bullous pemphigoid, pemphigus vulgaris), but not in normal interfollicular epidermis. In contrast to normal keratinocytes, HaCaT cells showed constitutive L6 expression, indicating a constitutively active phenotype. After artificial wounding of confluent HaCaT cultures, anti-L6 antibody strongly impaired cell migration velocity and migratory reepithelization of the defect, indicating L6 involvement in keratinocyte migration. These findings suggest that L6 is an important activation-dependent regulator of keratinocyte function and epidermal tissue regeneration.

Inhibitory effects of 17beta-estradiol and progesterone on ovarian carcinoma cell proliferation: a potential role for inducible nitric oxide synthase.

OBJECTIVES: Indirect evidence suggests that estrogen and progesterone are involved in the etiology of ovarian cancer (Oca). Estrogen and progesterone are also thought to modulate nitric oxide (NO) in human ovarian tumor tissue via regulation of inducible nitric oxide synthase (iNOS). Objectives in this study were: (1) to investigate the effects of 17beta-estradiol (E(2)) and progesterone (P(4)) on Oca cell proliferation employing elevated hormone concentrations occurring within the microenvironment of the ovary, and (2) to determine whether E(2) or P(4) affects iNOS expression and NO generation in Oca cells. METHODS: Proliferation assays assessed the effects of E(2) and P(4) on cell growth in three human Oca cell lines (HOC-7, OVCAR-3, SKOV-3). Reverse transcriptase polymerase chain reaction was used to assess mRNA expression and Western blots to determine protein levels. NO generation was determined via the Griess reaction. RESULTS: Elevated E(2), P(4), or E(2) plus P(4) (E + P), significantly inhibited HOC-7 cells and OVCAR-3 cells, but not SKOV-3 cells. E(2) at 10 microM downregulated iNOS expression and significantly reduced NO production in HOC-7 cells, while 10 microM P(4) or 10 microM E + P increased iNOS expression and NO production. Conclusions. Our findings demonstrate that elevated E(2), P(4), or E + P results in significant growth inhibition of Oca cells, and we propose a role for iNOS and NO in how these hormones modulate their activities in Oca cells.

Antisense oligonucleotide suppression of Na(+)/Ca(2+) exchanger activity in primary neurons from rat brain.

An antisense (AS) oligodeoxynucleotide based on a conserved sequence in the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) was used to decrease expression of this Ca(2+) transporter in primary neuronal cultures. Two AS oligo applications decreased NCX activity by approximately 40% within 12-24 h, and neither sense (S) or missense (MS) oligos altered NCX activity. The reduced NCX expression was confirmed by immunoblots and enzyme-linked immunosorbent assays (ELISAs). Resting [Ca(2+)](i) levels were 20% higher in AS-treated neurons and showed a slower return to baseline levels following activation of Ca(2+) influx by N-methyl-D-aspartate (NMDA). These results suggest that NCX plays a significant role in maintaining neuronal Ca(2+) homeostasis and in restoring baseline Ca(2+) levels following depolarization.

Immunohistochemical and molecular characterization of cultured keratinocytes after dispase-mediated detachment from the growth substratum.

Keratinocyte activation comprises changes in protein and gene expression pattern resulting in phenotypic and functional changes necessary for re-epithelialization such as the expression of urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPA-R; CD87). As uPA and uPA-R are rapidly induced after dispase-mediated detachment of cultured normal human epidermal keratinocytes (NHEK) we hypothesized that dispase-mediated detachment may cause a similar "activation" of keratinocytes with uPA and uPA-R being only one aspect of a complex "activation reaction". To test this hypothesis we have comparatively analysed adherent versus detached keratinocyte sheets for selected indicators of keratinocyte activation by immunohistochemistry. Furthermore we have identified genes via subtraction cloning which are up-regulated upon dispase-induced detachment. The analyses provided evidence for an increased transcriptional and translational activity in detached keratinocytes, as indicated by over-expression of several ribosomal components (L3 and S10 ribosomal protein) and transcription factors (initiation factor 4A, elongation factor 1alpha). Increased proliferative activity was indicated by increased expression of the proliferation markers Ki67, keratin 6 and keratin 17. Finally, several markers of keratinocyte activation such as the integrin chain alpha(v), psoriasin, glutathion-S-transferase and heparin-binding epidermal growth factor-like growth factor were up-regulated. Furthermore mevalonate kinase, a molecule as yet unknown to be expressed in keratinocytes, was identified. The findings provide evidence that dispase-mediated detachment in cultured keratinocytes induces a reaction, which comprises the up-regulation of a complex array of proliferation- and migration-related molecules. The pattern of which resembles the activation reaction observed in the re-epithelializing keratinocytes in vivo.

Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.

Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula.

Pathogenicity induced by feline leukemia virus, Rickard strain, subgroup A plasmid DNA (pFRA).

A new provirus clone of feline leukemia virus (FeLV), which we named FeLV-A (Rickard) or FRA, was characterized with respect to viral interference group, host range, complete genome sequence, and in vivo pathogenicity in specific-pathogen-free newborn cats. The in vitro studies indicated the virus to be an ecotropic subgroup A FeLV with 98% nucleotide sequence homology to another FeLV-A clone (F6A/61E), which had also been fully sequenced previously. Since subgroup B polytropic FeLVs (FeLV-B) are known to arise via recombination between ecotropic FeLV-A and endogenous FeLV (enFeLV) env elements, the in vivo studies were conducted by direct intradermal inoculation of the FRA plasmid DNA so as to eliminate the possibility of coinoculation of any FeLV-B which may be present in the inoculum prepared by propagating FeLV-A in feline cell cultures. The following observations were made from the in vivo experiments: (i) subgroup conversion from FeLV-A to FeLV-A and FeLV-B, as determined by the interference assay, appeared to occur in plasma between 10 and 16 weeks postinoculation (p.i.); (ii) FeLV-B-like recombinants (rFeLVs), however, could be detected in DNA isolated from buffy coats and bone marrow by PCR as early as 1 to 2 weeks p.i.; (iii) while a mixture of rFeLV species containing various amounts of N-terminal substitution of the endogenous FeLV-derived env sequences were detected at 8 weeks p.i., rFeLV species harboring relatively greater amounts of such substitution appeared to predominate at later infection time points; (iv) the deduced amino acid sequence of rFeLV clones manifested striking similarity to natural FeLV-B isolates, within the mid-SU region of the env sequenced in this work; and (v) four of the five cats, which were kept for determination of tumor incidence, developed thymic lymphosarcomas within 28 to 55 weeks p.i., with all tumor DNAs harboring both FeLV-A and rFeLV proviruses. These results provide direct evidence for how FeLV-B species evolve in vivo from FeLV-A and present a new experimental approach for efficient induction of thymic tumors in cats, which should be useful for the study of retroviral lymphomagenesis in this outbred species.

In vivo evolution and selection of recombinant feline leukemia virus species.

Ecotropic feline leukemia viruses subgroup A (FeLV-A) is known to recombine with endogenous FeLV (enFeLV) env elements yielding polytropic FeLV-B viruses. However, scattered nucleotide differences exist between enFeLV env elements and corresponding sequences of exogenous FeLV-B isolates. To address this disparity, we examined recombinant FeLV (rFeLV) viruses obtained from three experimentally-induced feline thymic tumors, along with rFeLVs derived from one naturally-occurring thymic tumor. Two of the three experimental cats were challenged with a FeLV-A/Rickard preparation, while one cat received this FeLV-A along with a mixture of in vitro-generated rFeLVs. The FeLV-A/Rickard preparation employed in this study was shown to be free of detectable rFeLVs since no recombinant products were observed in this preparation following nested PCR analyses. For each of the four tumor DNAs, nucleotide sequence analysis was performed on multiple clones of rFeLV-specific PCR products derived from the surface glycoprotein (SU) portion of the recombinant proviral env gene. Relative to the parental enFeLV sequence used to generate the rFeLVs, a total of 19 nucleotide differences were found scattered within the SU region of the env gene in these in vivo-derived rFeLV clones. Most interestingly, this set of 19 differences led to complete sequence identity with natural FeLV-B isolates. Our results indicate these differences are present early in the in vivo evolution of recombinant viruses, suggesting that rFeLVs harboring these differences are strongly selected. We also present evidence indicating an in vivo selection pattern exists for specific recombinant species containing relatively greater amounts of enFeLV-derived SU sequence. This in vivo selection process appears to be gradual, occurring over the infection timecourse, yielding rFeLV species which have recombination structural motifs similar to those seen in natural FeLV-B isolates.

Protection against beta-amyloid toxicity in primary neurons by paclitaxel (Taxol).

Neurofibrillary tangles in Alzheimer's disease contain aggregates of abnormally phosphorylated microtubule-associated protein tau, indicating that microtubule breakdown is a primary event in the neurodegenerative cascade. Recent studies have shown that addition to neuronal cultures of amyloid peptides found in Alzheimer's leads to abnormal phosphorylation of tau and neurofibrillary pathology. We tested the possibility that the microtubule-stabilizing drug paclitaxel (Taxol) might protect primary neurons against amyloid-induced toxicity. Neurons exposed to aggregated amyloid peptides 25-35 and 1-42 became pyknotic with degenerating neurites within 24 h. Treatment of cultures with paclitaxel either 2 h before or 2 h after addition of the peptide prevented these morphological alterations. When numbers of viable cells were determined in cultures exposed to amyloid peptide with or without paclitaxel for 24 or 96 h, the percentage of surviving cells was significantly higher in paclitaxel-treated cultures, and activation of the apoptosis-associated protease CPP32 was significantly reduced. These observations indicate that microtubule-stabilizing drugs may help slow development of the neurofibrillary pathology that leads to the loss of neuronal integrity in Alzheimer's disease.

Plasminogen activator inhibitor type 2 is expressed in keratinocytes during re-epithelialization of epidermal defects.

Plasminogen activation is observed in the human epidermis during re-epithelialization of epidermal defects. The activation reaction depends on plasminogen activators (PAs) associated with re-epithelializing keratinocytes. PA inhibitor type 2 (PAI-2) is thought to be a major epidermal PA inhibitor in keratinocytes. However, no data are available on the expression of PAI-2 in keratinocytes during epidermal regeneration. We have therefore analysed PAI-2 at the mRNA and protein level in keratinocyte cultures as well as in epidermal lesions in which re-epithelializing keratinocytes were apparent. We found that PAI-2 expression at the mRNA and protein level was negatively correlated with the cell density in regular keratinocyte cultures. In organotypic cocultures, in which the transition from a re-epithelializing to a sedentary phenotype can be studied, PAI-2 was most strongly expressed in early cultures prior to formation of a differentiated epidermis-like structure. We found a strong expression of PAI-2 in keratinocytes that re-epithelialized dermal burn wounds or lesions caused by the autoimmune blistering disease pemphigus vulgaris. Our results suggest that not only PAs, but also a major PA inhibitor, PAI-2, are expressed in keratinocytes that are actively involved in re-epithelialization.

Dispase-mediated basal detachment of cultured keratinocytes induces urokinase-type plasminogen activator (uPA) and its receptor (uPA-R, CD87).

Keratinocytes synthesize and secrete urokinase-type plasminogen activator (uPA), which is bound in an autocrine manner to a specific receptor (uPA-R, CD87) at their surface. Plasminogen, which is also bound to membrane binding sites, is readily activated by uPA-R-bound uPA. Thus, plasmin for proteolysis of pericullular glycoproteins is provided. While uPA-R and uPA are at low to undetectable levels in keratinocytes of the normal epidermis, both compounds are upregulated in migrating keratinocytes during reepithelialization of epidermal defects and in affected keratinocytes of various epidermal disorders, including bullous dermatoses. We have hypothesized that the disturbance of cell/matrix interactions--a common feature of these diverse pathological situations--induces uPA/uPA-R. Accordingly, we explored whether the dispase-mediated detachment of cultured keratinocytes, which have formed a multilayered epidermis-like structure in vitro, induced uPA and uPA-R. We found increases in uPA secretion, cell-associated uPA activity, and uPA- and uPA-R-antigen in keratinocytes upon dispase-mediated detachment from their growth substratum. The increase was preceded by an increase in uPA-R- and uPA-specific mRNA, which was not observed when the proteinase inhibitor phosphoramidon was added together with dispase. In conclusion, we present evidence that experimental detachment with dispase provides signals for the concomitant upregulation of uPA-R and uPA. The findings support the hypothesis that cell/matrix interactions may influence the expression of the cell surface-associated PA system in human keratinocytes.