Fluid shear promotes chondrosarcoma cell invasion by activating matrix metalloproteinase 12 via IGF-2 and VEGF signaling pathways.

Interstitial fluid flow in and around the tumor tissue is a physiologically relevant mechanical signal that regulates intracellular signaling pathways throughout the tumor. Yet, the effects of interstitial flow and associated fluid shear stress on the tumor cell function have been largely overlooked. Using in vitro bioengineering models in conjunction with molecular cell biology tools, we found that fluid shear (2 dyn/cm(2)) markedly upregulates matrix metalloproteinase 12 (MMP-12) expression and its activity in human chondrosarcoma cells. MMP-12 expression is induced in human chondrocytes during malignant transformation. However, the signaling pathway regulating MMP-12 expression and its potential role in human chondrosarcoma cell invasion and metastasis have yet to be delineated. We discovered that fluid shear stress induces the synthesis of insulin growth factor-2 (IGF-2) and vascular endothelial growth factor (VEGF) B and D, which in turn transactivate MMP-12 via PI3-K, p38 and JNK signaling pathways. IGF-2-, VEGF-B- or VEGF-D-stimulated chondrosarcoma cells display markedly higher migratory and invasive potentials in vitro, which are blocked by inhibiting MMP-12, PI3-K, p38 or JNK activity. Moreover, recombinant human MMP-12 or MMP-12 overexpression can potentiate chondrosarcoma cell invasion in vitro and the lung colonization in vivo. By reconstructing and delineating the signaling pathway regulating MMP-12 activation, potential therapeutic strategies that interfere with chondrosarcoma cell invasion may be identified.

Loss of giant obscurins from breast epithelium promotes epithelial-to-mesenchymal transition, tumorigenicity and metastasis.

Obscurins, encoded by the single OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. The OBSCN gene is highly mutated in different types of cancers. Loss of giant obscurins from breast epithelial cells confers them with a survival and growth advantage, following exposure to DNA-damaging agents. Here we demonstrate that the expression levels and subcellular distribution of giant obscurins are altered in human breast cancer biopsies compared with matched normal samples. Stable clones of non-tumorigenic MCF10A cells lacking giant obscurins fail to form adhesion junctions, undergo epithelial-to-mesenchymal transition and generate >100-µm mammospheres bearing markers of cancer-initiating cells. Obscurin-knockdown MCF10A cells display markedly increased motility as a sheet in 2-dimensional (2D) substrata and individually in confined spaces and invasion in 3D matrices. In line with these observations, actin filaments redistribute to extending filopodia where they exhibit increased dynamics. MCF10A cells that stably express the K-Ras oncogene and obscurin short hairpin RNA (shRNA), but not scramble control shRNA, exhibit increased primary tumor formation and lung colonization after subcutaneous and tail vein injections, respectively. Collectively, our findings reveal that loss of giant obscurins from breast epithelium results in disruption of the cell-cell contacts and acquisition of a mesenchymal phenotype that leads to enhanced tumorigenesis, migration and invasiveness in vitro and in vivo.

Presoaking with BSS used for thin manually dissected DSEK (TMDSEK): a viable option for thin DSEK.

AIM: To describe a novel technique for the safe manual dissection of thin donor lenticules in 10 consecutive patients undergoing DSEK surgery. METHODS: A key element of our new technique was to presoak the donor cornea in balanced salt solution (BSS) for 30 min before manual dissection. The cornea was placed on an artificial anterior chamber and pressure in the chamber was maintained at 80 mm Hg. The limbus of the donor cornea was incised to the same depth as the central corneal thickness. Lamellar dissection was started with the short side of the Morlet dissector (Duckworth & Kent Ltd) and completed using the lamellar (less sharp) end of the Morlet dissector. Outcomes of 10 consecutive cases of thin manually dissected DSEK (TMDSEK) were prospectively analysed for thickness and visual outcome. RESULTS: Mean graft thicknesses measured less than 100 µm at 1 month post surgery (mean thickness 90.7 µm, range 48-137 µm, SD 29.96 µm). Presoaked donor corneal pachymetry was strongly negatively correlated with graft thickness (correlation r=-0.75, P<0.05). DISCUSSION: Our dissection technique achieves consistently thin endothelial donor corneal grafts that can be safely produced with minimal financial investment and no limitations on surgical time. This technique is likely to be of significant importance for a large proportion of the eye centres where microkeratomes may not be routinely available.

Prostaglandin (PG)D(2) and 15-deoxy-Delta(12,14)-PGJ(2), but not PGE(2), mediate shear-induced chondrocyte apoptosis via protein kinase A-dependent regulation of polo-like kinases.

Excessive mechanical loading of cartilage producing hydrostatic stress, tensile strain and fluid flow leads to chondrocyte apoptosis and osteoarthritis. High fluid flow induces cyclooxygenase-2 (COX-2) expression in sheared chondrocytes, which suppresses their antioxidant capacity and contributes to apoptosis. The pivotal role of COX-2 in shear-induced chondrocyte apoptosis and the conflicting literature data on the roles of prostaglandin (PG)E(2), PGD(2) and its metabolite 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in chondrocyte apoptosis prompted us to analyze which COX-2-derived PG is involved in this process. We show that exogenously added PGD(2) and 15d-PGJ(2), but not PGE(2), diminish the viability of human T/C-28a2 chondrocytes under static conditions. In agreement with these observations, knockdown of L-PGD synthase (L-PGDS) abolishes shear-induced chondrocyte apoptosis. Using cDNA microarrays in conjunction with clustering algorithms, we propose a novel signaling pathway by which high fluid shear mediates COX-2/L-PGDS-dependent chondrocyte apoptosis, which is validated by molecular interventions. We show that L-PGDS controls the downregulation of protein kinase A (PKA), which in turn regulates Polo-like kinase1 (Plk1) and Plk3. Plks target p53, which controls the transcription of p53 effectors (TP53INPs, FAS and Bax) involved in chondrocyte apoptosis. Reconstructing the signaling network regulating chondrocyte apoptosis may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic strategies for arthritic disorders.

Seroepidemiology of hepatitis A among Greek children indicates that the virus is still prevalent: Implications for universal vaccination.

A national cross-sectional seroprevalence survey was conducted in order to evaluate the current seroepidemiology of hepatitis A among 1,383 children, aged 0-14 years, residing in Greece. Stratification of the study population was conducted according to age and area of residence. Sera from study participants were tested for the presence of anti-HAV IgG antibodies. Immigrant children, as well as children residing in rural areas, had lower immunization rates. Among unvaccinated children, the seroprevalence rate of anti-HAV was 17.1%. Nationality was shown to have a marginally significant effect since non-immunized immigrant children had a higher seroprevalence rate (22.4% vs. 15.9%, OR = 1.52, P = 0.064). Significant differences between geographic areas for both vaccination coverage and natural immunity were observed. The study findings indicate that hepatitis A is prevalent in Greece and therefore universal infant hepatitis A immunization should be implemented.

Outcome of intravitreal triamcinolone acetonide in postoperative cystoid macular oedema.

AIMS: To assess the efficacy and safety of intravitreal triamcinolone in the treatment of postoperative cystoid macular oedema (CMO). METHODS: A retrospective case series review of 21 eyes (20 patients) that had an intravitreal injection of triamcinolone 4 mg for postoperative CMO. Diagnosis was confirmed by fundus fluorescein angiography and/or optical coherence tomography in all eyes. RESULTS: Mean age of patients was 71.1 years. CMO had developed following routine phacoemulsification cataract extraction (13 eyes), phacoemulsification cataract extraction complicated by posterior capsule tear and vitreous loss (two eyes), vitrectomy (three eyes), or planned combined phacoemulsification and vitrectomy (three eyes). Mean duration of CMO before triamcinolone injection was 4.9 months. Mean duration of follow-up was 7.4 months. Two eyes required a repeat injection. Mean logarithm of minimum angle of resolution (LogMAR) visual acuity (VA) before treatment was 0.53; at 1 month post injection, this increased significantly to 0.33 (P<0.001). Improvement in VA was maintained throughout follow-up; at 6 months or later, mean LogMAR VA was significantly better than baseline (0.33 vs 0.53, P=0.02). At the latest review, 43% of eyes had improved Snellen VA by two or more lines and 86% by one or more lines compared to baseline. The remaining 14% had reduced Snellen VA compared to baseline. In the post-injection period, 33% of eyes developed an intraocular pressure of 22 mm Hg or higher and all responded well to short-term topical agents. There were no other post-injection complications. CONCLUSION: Intravitreal triamcinolone results in a rapid improvement in VA that may be sustained for more than 6 months.

Near work, education, family history, and myopia in Greek conscripts.

AIMS: To investigate potential factors associated with the presence of myopia in a cohort of young adult men carrying out their military service in Greece. METHODS: A nested case-control study of 200 conscripts (99 myopes and 101 non-myopes). The cohort consisted of approximately 1000 conscripts in compulsory national service. All cohort members had been screened for refractive errors by Snellen visual acuity measurement at presentation to military service; individuals not achieving visual activity 6/6 underwent noncycloplaegic refraction. The study sample consisted of the first 99 myopic and 101 nonmyopic conscripts who attended the study. In-person interviews of these 200 conscripts were conducted to obtain information on family history, occupation, level of education, near-work activities, and sleeping behaviour. chi(2) and Mann-Whitney tests were used as univariate analysis methods to identify the potential factors associated with the presence of myopia. Multiple logistic regression was used to estimate the adjusted relative risk of myopia. RESULTS: Univariate analysis showed that parental family history (P<0.001), older age (P<0.001), tertiary education (P<0.001), hours of reading per day (P<0.001), hours of computer use per day (P<0.001), and higher social classes (P<0.001) were associated with myopia. Sleeping in artificial or ambient light was not associated with myopia (P=0.75). Multiple logistic regression analysis showed that older age (OR=1.25, 95% CI 1.05-1.49), tertiary education (OR=12.67, 95% CI 3.57-44.88) and parental family history (OR=3.39, 95% CI 1.56-7.36) were independently associated with myopia. CONCLUSION: In young Greek conscripts, parental family history, older age, and education level are independently associated with myopia.

Meningococcal group C disease in Greece during 1993-2006: the impact of an unofficial single-dose vaccination scheme adopted by most paediatricians.

The aim of this study was to evaluate the impact of the meningococcal C conjugate vaccine on the epidemiology of meningococcal C disease in Greece. Data from the National Reference Laboratory for Meningococcal Disease and a questionnaire distributed to Greek paediatricians were assessed. Since the introduction of the vaccine in 2001, 72% of Greek paediatricians have administered it as one single dose to patients aged > or =12 months. This vaccination scheme has probably contributed to a dramatic decrease in the number of meningococcal C infections, which reached zero in 2004.

Ocular morbidity associated with intravitreal triamcinolone acetonide.

AIM: To report on the complications associated with the use of intravitreal triamcinolone acetonide (IVTA) in a tertiary referral hospital setting. MATERIALS AND METHODS: A retrospective case series review of all IVTA injections carried out over a period of 30 months. RESULTS: One hundred and thirty IVTA injections were performed; nine with limited local follow-up were excluded. Thus, 121 injections (108 patients, 114 eyes) were included in the study. Triamcinolone (4 mg) was used in all cases. Indications were diabetic macular oedema (n=41 eyes), retinal vein occlusions (n=27), postoperative cystoid macular oedema (n=24), exudative age-related macular degeneration (n=16), and others (n=6). No intraoperative complications were recorded. Postoperative intraocular pressure (IOP) readings of 22, 28, 35, and 40 mmHg or higher were recorded in 46.5, 29.8, 12.3, and 7.9% of eyes, respectively. IOP elevation was treated with antiglaucoma medication in all but one eye (0.9%) that required trabeculectomy and one (0.9%) that required vitrectomy with cataract extraction for suspected phacoanaphylactic glaucoma. Two eyes (1.8%) developed retinal detachment; both had previously been treated for retinal breaks. One eye (0.9%) developed culture-positive endophthalmitis. CONCLUSIONS: Significant morbidity is associated with IVTA injection; clinicians should be aware when considering treatment options.

Evaluation of a quick test for C-reactive protein in a pediatric emergency department.

OBJECTIVE: C-reactive protein (CRP) is a reliable laboratory test that is useful in distinguishing between viral and bacterial infection. Although widely used, blood sampling and the need for a well-organized laboratory are limiting factors. Recently, a rapid test for serum CRP (QuickRead CRP) has been developed that can use both venous and capillary blood. The aim of this study was to use QuickRead CRP in our Pediatric Emergency Department and to compare this method with the standard laboratory determination (CRP-lab). MATERIAL AND METHODS: All children with fever were given a quick CRP test simultaneously with venous (CRP-V) and capillary blood samples (CRP-C). A total of 127 children were included in the study (median age 2.5 years). RESULTS: The QuickRead CRP test had an excellent correlation with the standard biochemical determination (CRP-lab). More importantly, there was no difference in determination of CRP in the venous and capillary blood samples. Finally, there was no significant intra-assay variability. CONCLUSIONS: The QuickRead CRP test is easy to use, provides reliable results and reduces the need for antibacterial therapy.

Invasive meningococcal disease in children in Greece: comparison of serogroup A disease with disease caused by other serogroups.

Although invasive meningococcal disease caused by serogroup A is not prevalent in developed countries, a considerable number of cases were recently recorded in Greece. In this study, serogroup A meningococcal disease was compared prospectively with meningococcal disease caused by other serogroups, using similar settings of testing and management during a 5-year period between 1999 and 2003. The Neisseria meningitidis serogroup was determined in 262 cases. Serogroup B predominated, accounting for 158 (60%) of the cases. Serogroup A was second most frequent (19%), followed by serogroups W135 (11%), C (8%), and Y (2%). No cases due to serogroup C were recorded during the last year of the study. Patients with serogroup A disease were older and had a milder course compared to patients infected with serogroups B or C. Toxic appearance, purpura, thrombocytopenia, abnormal coagulation tests, and the need for admission to the intensive care unit, fluid resuscitation, inotropic drugs, and mechanical ventilation were less common. Although morbidity and mortality were lower in these patients, the differences were not significant. Serogroup B is predominant in our area, and the introduction of an effective vaccine against it is a priority. Serogroup A has emerged as the second most common serogroup, but the illness associated with it is milder.

Impact of influenza infection in healthy children examined as outpatients and their families.

Decisions regarding the introduction of influenza immunization in healthy children require an accurate evaluation of influenza disease burden not only in the inpatient but also in the outpatient setting. We prospectively examined the impact of virologically confirmed influenza in 1462 outpatient children (> or = 6 months to < 14 years) and their families, during two consecutive influenza seasons. Influenza was documented in 573/1462 (39%) outpatients with febrile respiratory illness and accounted for 13.5% of all outpatient visits during the 14 weeks of each season. Acute otitis media (AOM) was the most common complication (18.5%) and about 40% of influenza positive patients received antibiotics. AOM and antibiotic use were more common in children younger than 5 years of age who accounted for 58% of all patients. For each child sick with influenza a mean of 1.34 workdays were lost by the parents. Family members of influenza positive children were more likely to develop a secondary respiratory illness and to require medical visits and antibiotic prescriptions. Influenza is associated with a heavy morbidity burden in the community that may be reduced considerably with the implementation of immunization in children younger than 5 years of age.

Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos.

SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.

The prototypical 4.1R-10-kDa domain and the 4.1g-10-kDa paralog mediate fodrin-actin complex formation.

A complex family of 4.1R isoforms has been identified in non-erythroid tissues. In this study we characterized the exonic composition of brain 4.1R-10-kDa or spectrin/actin binding (SAB) domain and identified the minimal sequences required to stimulate fodrin/F-actin association. Adult rat brain expresses predominantly 4.1R mRNAs that carry an extended SAB, consisting of the alternative exons 14/15/16 and part of the constitutive exon 17. Exon 16 along with sequences carried by exon 17 is necessary and sufficient to induce formation of fodrin-actin-4.1R ternary complexes. The ability of the respective SAB domains of 4.1 homologs to sediment fodrin/actin was also investigated. 4.1G-SAB stimulates association of fodrin/actin, although with an approximately 2-fold reduced efficiency compared with 4.1R-10-kDa, whereas 4.1N and 4.1B do not. Sequencing of the corresponding domains revealed that 4.1G-SAB carries a cassette that shares significant homology with 4.1R exon 16, whereas the respective sequence is divergent in 4.1N and absent from brain 4.1B. An approximately 150-kDa 4.1R and an approximately 160-kDa 4.1G isoforms are present in PC12 lysates that occur in vivo in a supramolecular complex with fodrin and F-actin. Moreover, proteins 4.1R and 4.1G are distributed underneath the plasma membrane in PC12 cells. Collectively, these observations suggest that brain 4.1R and 4.1G may modulate the membrane mechanical properties of neuronal cells by promoting fodrin/actin association.

A nonerythroid isoform of protein 4.1R interacts with components of the contractile apparatus in skeletal myofibers.

The approximately 80-kDa erythroid 4.1R protein is a major component of the erythrocyte cytoskeleton, where it links transmembrane proteins to the underlying spectrin/actin complexes. A diverse collection of 4.1R isoforms has been described in nonerythroid cells, ranging from approximately 30 to approximately 210 kDa. In the current study, we identified the number and primary structure of 4.1R isoforms expressed in adult skeletal muscle and characterized the localization patterns of 4.1R message and protein. Skeletal muscle 4.1R appears to originate solely from the upstream translation initiation codon (AUG-1) residing in exon 2'. Combinations of alternatively spliced downstream exons generate an array of distinct 4.1R spliceoforms. Two major isoform classes of approximately 105/110 and approximately 135 kDa are present in muscle homogenates. 4.1R transcripts are distributed in highly ordered signal stripes, whereas 4.1R protein(s) decorate the sarcoplasm in transverse striations that are in register with A-bands. An approximately 105/110-kDa 4.1R isoform appears to occur in vivo in a supramolecular complex with major sarcomeric proteins, including myosin, alpha-actin, and alpha-tropomyosin. In vitro binding assays showed that 4.1R may interact directly with the aforementioned contractile proteins through its 10-kDa domain. All of these observations suggest a topological model whereby 4.1R may play a scaffolding role by anchoring the actomyosin myofilaments and possibly modulating their displacements during contraction/relaxation.

Embryonic and post-embryonic utilization and subcellular localization of the nuclear receptor SpSHR2 in the sea urchin.

SpSHR2 (Strongylocentrotus purpuratus steroid hormone receptor 2) is a nuclear receptor, encoded by a maternal RNA in the sea urchin embryo. These maternal SpSHR2 transcripts, which are present in all cells, persist until the blastula stage and then are rapidly turned over. A small fraction of the embryonic SpSHR2 protein is maternal, but the majority of this nuclear receptor in the embryo is the product of new synthesis, presumably from the maternal RNA after fertilization. In agreement with the mRNA distribution, the SpSHR2 protein is also detected in all embryonic cells. Contrary to the RNA though, the SpSHR2 protein persists throughout embryonic development to the pluteus stage, long after the mRNA is depleted. Following fertilization and as soon as the 2-cell stage, the cytoplasmic SpSHR2 protein enters rapidly into the embryonic nuclei where it appears in the form of speckles. During subsequent stages (from fourth cleavage onward), SpSHR2 resides in speckled form in both the nucleus and the cytoplasm of the embryonic cells. The cytoplasmic localization of SpSHR2 differs between polarized and non-polarized cells, maintaining an apical position in the ectoderm and endoderm versus a uniform distribution in mesenchyme cells. Following the end of embryonic development (pluteus stage), the SpSHR2 protein is depleted from all tissues. During the ensuing four weeks of larval development, the SpSHR2 is not detected in either the larval or the rudiment cells which will give rise to the adult. Just prior to metamorphosis, at about 35 days post-fertilization, the protein is detected again but in contrast to the uniform distribution in the early embryo, the larval SpSHR2 is specifically expressed in cells of the mouth epithelium and the epaulettes. In adult ovaries and testes, SpSHR2 is specifically detected in the myoepithelial cells surrounding the ovarioles and the testicular acini. Nuclear SpSHR2 in blastula extracts binds to the C1R hormone response element in the upstream promoter region of the CyIIIb actin gene indicating that the latter may be a target of this nuclear receptor in the sea urchin embryo.

A novel sea urchin nuclear receptor encoded by alternatively spliced maternal RNAs.

Screening of a genomic library from the sea urchin Strongylocentrotus purpuratus with a human COUP-TF I cDNA probe revealed the presence of a novel gene member of the steroid-thyroid-retinoic acid receptor superfamily, which was named SpSHR2 (S. purpuratus Steroid Hormone Receptor 2). Sequence analysis of the isolated genomic clone revealed that the DNA binding domain of this orphan receptor is most homologous to the human TR2 receptor. Using this sea urchin genomic fragment as probe, a S. purpuratus embryonic cDNA library was screened and two distinct but homologous cDNA clones were isolated. The two cDNAs encode the same DNA binding domain as the SpSHR2 gene and carry an almost identical 3'-untranslated sequence. One of the clones, however, is missing an entire region of about 1100 nt which includes the putative ligand binding domain. Genomic DNA hybridization suggests that SpSHR2 is a single-copy gene in the S. purpuratus genome. Exon skipping during splicing of a single primary transcript appears to be the reason for the differently sized mRNAs. RNA blot hybridization results suggest that SpSHR2 transcripts are stored as maternal RNA in the egg and are not detected beyond the blastula stage. In vitro transcription and translation of the full-length cDNA produced a polypeptide which specifically binds to the hormone response element in the 5'-flanking region of the sea urchin actin CyIIIb gene. In vivo labeling of proteins synthesized by cleavage stage embryos followed by immune precipitation of the SpSHR2 protein using specific antibodies reveals that the maternal SpSHR2 mRNA is being translated during early embryonic development.