Physical Therapy Exercises for Sleep Disorders in a Rehabilitation Setting for Neurological Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Sleep occupies one-third of human life and is essential for health and for emotional, physical, and cognitive well-being. Poor or insufficient sleep is associated with a wide range of dysfunctions that involve different body systems, such as the endocrine, metabolic, and immune systems, thus compromising the higher cortical functions, cognitive performance, mood, and post-physical activity recovery. The present systematic review and meta-analysis aimed to explore the effectiveness of physical therapy exercises on sleep disorders in patients with neurological disorders. Our systematic review identified 10 articles that investigated the effects of physical therapy on sleep disorders in patients with neurological disorders, 6 of which were included in the meta-analysis. Results suggest that physical therapy exercises are a safe and useful strategy for managing sleep disorders in neurorehabilitation.

Silencing of telomere-binding protein adrenocortical dysplasia (ACD) homolog enhances radiosensitivity in glioblastoma cells.

Adrenocortical dysplasia (ACD) is a shelterin protein involved in the maintenance of telomere length and in cancer radioresistance. This study investigated the expression profile of ACD in human gliomas and its role in radioresistance of glioma cells. The expression of ACD was analyzed in 62 different grades of glioma tissues and correlated with prognosis. A radioresistant cell line was generated from U87MG cells. For mechanistic studies, ACD was inhibited by small interfering RNA-targeting ACD and the effect on cell radioresistance, telomerase activity, cyclinD1, caspase-3, hTERT, and BIRC1 was evaluated. Clonogenic assay was performed after irradiation, to investigate the effect of ACD silencing on radiation sensitivity. ACD expression appeared strongly upregulated in higher grade gliomas, and its expression was significantly correlated to grading and poor prognosis. In glioma cell lines, ACD expression pattern was similar to those observed in glioma tissues. In irradiated cells, ACD expression was increased in an ionizing radiation dose-dependent manner. A higher expression of ACD was observed in the radioresistant clones than parental cells. Silencing of ACD led to the enhanced radiation sensitivity, decreased telomerase activity and cyclin D1 expression, reduced expression of BIRC1, and finally to the upregulation of caspase-3. This study represents the first report, which demonstrated the expression pattern of ACD in gliomas and its prognostic value. Our results suggested that ACD is involved in glioblastoma radioresistance, likely through the modulation of telomerase activity, proliferation, and apoptosis. ACD might represent a potential molecular biomarker and a novel therapeutic target in glioblastoma.

Ischemic stroke due to hypoperfusion in a patient with a previously unrecognized Danon disease.

Danon disease, an X-linked multisystemic disorder, is due to deficiency of Lysosome-Associated Membrane Protein 2 (LAMP2). It is usually characterized by hypertrophic cardiomyopathy, mental retardation and skeletal myopathy, sometimes also with atypical features. A 20-year-old man with cognitive impairment was admitted to the Emergency Room because of a sudden chest pain. ECG showed Wolff-Parkinson-White syndrome; echocardiography revealed hypertrophic cardiomyopathy, and, shortly after, he experienced a cardiac arrest followed by an occipital ischemic stroke. On neurological examination, he complained of visual loss, and diffuse muscle wasting and weakness were also unexpectedly noted. Electromyography evidenced a myopathic pattern and a peripheral neuropathy. A muscle biopsy disclosed vacuolar myopathy with glycogen storage; immunohistochemical studies demonstrated a LAMP-2 deficiency. LAMP2 molecular analysis identified a "de novo" mutation (p. Q353X). This patient with a neglected Danon disease, experienced an unusual complication as a stroke due to cerebral hypoperfusion after cardiac arrest caused by WPW syndrome.

miRNA regulation of Sirtuin-1 expression in human astrocytoma.

Sirtuins are a family of 7 histone deacetylases largely involved in the regulation of cell proliferation, survival and death. The role of sirtuins in tumorigenesis and cancer progression has been previously studied in certain cancer types. Few studies have investigated sirtuin expression in gliomas, with controversial results. The aim of the present study was to investigate the expression of sirtuin-1 (Sirt-1) in diffuse astrocytoma [low grade astrocytoma (LGA)], anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) and in primary glioma cell lines: PLGAC (primary LGA cells); PAAC (primary AA cells); and PGBMC (primary GBM cells). Tumor samples were obtained from patients who underwent craniotomy for microsurgical tumor resection at the Neurosurgery Unit of the University of Messina between 2011 and 2014. Sirt-1 expression was qualitatively analyzed in 30 human glial tumor samples and 5 non-neoplastic brain tissue (NBT) specimens using immunohistochemistry and western blotting techniques. Sirt-1 expression was quantitatively analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, Sirt-1 expression in primary cell lines was investigated by immunoblotting and RT-qPCR. Sirt-1 expression was downregulated in gliomas compared to NBTs. Sirt-1 levels also varied among different tumor grades, with more evident downregulation in high-grade (P<0.001) than low-grade tumors (P<0.01). These data were confirmed in cell lines, with the exception of upregulation of protein level in the highest malignancy grade cell lines. The present results suggest a role for miRNA-34a, miRNA-132 and miRNA-217 in the epigenetic control of Sirt-1 during gliomagenesis and progression, and demonstrate the different implications of Sirt-1 in human tissues and cell lines. Furthermore, the present results reveal that Sirt-1 may be an intrinsic regulator of tumor progression and the regulation of Sirt-1 involves complex molecular pathways. However, the biological functions of Sirt-1 in gliomagenesis require additional investigation.

Meningiomas and Proteomics: Focus on New Potential Biomarkers and Molecular Pathways.

Meningiomas are one of the most common tumors affecting the central nervous system, exhibiting a great heterogeneity in grading, treatment and molecular background. This article provides an overview of the current literature regarding the molecular aspect of meningiomas. Analysis of potential biomarkers in serum, cerebrospinal fluid (CSF) and pathological tissues was reported. Applying bioinformatic methods and matching the common proteic profile, arising from different biological samples, we highlighted the role of nine proteins, particularly related to tumorigenesis and grading of meningiomas: serpin peptidase inhibitor alpha 1, ceruloplasmin, hemopexin, albumin, C3, apolipoprotein, haptoglobin, amyloid-P-component serum and alpha-1-beta-glycoprotein. These proteins and their associated pathways, including complement and coagulation cascades, plasma lipoprotein particle remodeling and lipid metabolism could be considered possible diagnostic, prognostic biomarkers, and eventually therapeutic targets. Further investigations are needed to better characterize the role of these proteins and pathways in meningiomas. The role of new therapeutic strategies are also discussed.

Prognostic value of HMGB1 and oxidative stress markers in multiple trauma patients: A single-centre prospective study.

Serious multiple traumatic injuries may rapidly become fatal or be complicated by a life-threatening sequelae leading to a significant increase of the mortality rate. Trauma scoring systems are used to evaluate the critical status of the patient and recently many different biomarkers have been taken into account to better estimate the potential clinical outcome. The aim of the present study is to analyse the expression pattern of high-mobility group box-1 (HMGB1), oxidative stress markers and nuclear factor erythroid 2-related (Nrf2) in critically ill traumatic patients (at hospital admittance and after 6 and 24 h), in order to find out their potential role as early post-traumatic predictors markers. Forty-seven patients admitted for multiple trauma and 15 healthy participants were prospectively recruited. Eight patients (17%) died within 92 h of admission; this subgroup of patients presented the highest severity scores and their HMGB1 expression levels were significantly correlated with ISS, whereas patients with higher ISS exhibited higher levels of HMGB1 (P <0.001). Our study suggests the role of HMGB1 as a predictive biomarker of outcome in injured patients and hypothesizes the protective role of Nrf2 in bringing down the oxidative stress and HMGB1 release; measuring HMGB1 in combination with Nrf2 might represent a potentially useful tool in the early detection of post-trauma complications.

Intracranial arterial abnormalities in patients with late onset Pompe disease (LOPD).

BACKGROUND AND OBJECTIVES: Pompe disease is a rare metabolic disorder due to lysosomal alpha-glucosidase (GAA) deficiency. It is considered as a multi-systemic disease since, although glycogen accumulation is largely prominent in heart, skeletal and respiratory muscles, other organs can also be affected. As regards the vascular system, few reports have documented cerebrovascular malformations in Pompe patients. The aim of this study was to define the presence and type of intracranial arterial abnormalities in a cohort of late onset Pompe disease (LOPD) patients. METHODS: We have studied 21 LOPD patients with cerebral CT angiography (CTA), using maximum intensity projection and volume rendering technique for 3D-image reconstruction. RESULTS: We found intracranial arterial abnormalities in 13/21 patients (62 %), of whom: 2/21 patients (9.5 %) showed an unruptured intracranial aneurysm (respectively 2 and 4 mm), 10/21 (47 %) had a vertebrobasilar dolichoectasia (VBD) and 1/21 a basilar artery fenestration. Signs of lacunar encephalopathy (insular, capsular and frontal subcortical lesions) were detected in 13/21 patients (62 %) and this correlated with the presence of respiratory impairment (p = 0.017). CONCLUSIONS: These findings differ from what has been previously observed in healthy, aged-matched populations and confirm that cerebral arteries abnormalities, mainly involving the posterior circle, are not so rare in LOPD patients and are often accompanied by a lacunar encephalopathy that might represent a hypoxic-ischemic origin. A CTA or an MRA is recommended, in LOPD patients, for early detection of cerebrovascular malformations as they could lead to life-threatening events such as sub-arachnoid haemorrhage or brainstem compression.

Clinical and pathophysiological clues of respiratory dysfunction in late-onset Pompe disease: New insights from a comparative study by MRI and respiratory function assessment.

Respiratory insufficiency commonly develops in patients with Late Onset Pompe Disease (LOPD). It is conceivable that a timely starting of enzyme replacement therapy could avoid this life-threatening complication. Respiratory function in LOPD is commonly evaluated with standard pulmonary tests which do not extensively provide an accurate definition of the muscular pathophysiology. In eleven patients with LOPD and five healthy subjects, we compared pulmonary function results with MRI data, based on scans of the right lung acquired on maximum expiration and inspiration. We observed that variations in the cranio-caudal lung height and of lung areas in inspiration and expiration (lung delta) as well as the area of diaphragmatic movement strongly correlated with pulmonary function results. Moreover, MRI data confirmed that development of respiratory insufficiency in LOPD is mainly due to the diaphragmatic weakness with sparing of the antero-posterior chest expansion related to the activity of the intercostal muscles. These results suggest that respiratory muscle MRI is a quick, useful and reproducible tool for patient management as well as a reliable outcome measure for future LOPD therapeutic trials.

NOD2 triggers PGE2 synthesis leading to IL-8 activation in Staphylococcus aureus-infected human conjunctival epithelial cells.

We previously showed that Staphylococcus aureus and Pseudomonas aeruginosa stimulate IL-8 expression in human conjunctival epithelial cells through different signal transduction pathways. As in some cell types both the bacteria may induce the release of prostaglandin E2 (PGE2) and PGE2 may affect the expression of IL-8, we aimed at investigating whether in human conjunctival cells infected with S. aureus or P. aeruginosa the activation of IL-8 transcription was mediated by PGE2 and which were the underlying molecular mechanisms. We found that S. aureus, but not P. aeruginosa, triggered IL-8 activation by increasing COX-2 expression and PGE2 levels in a time-dependent manner. Overexpression of nucleotide-binding oligomerization domain-2 (NOD2) resulted to be essential in the enhancement of IL-8 induced by S. aureus. It dramatically activated c-jun NH2-terminal kinase (JNK) pathway which in turn led to COX2 upregulation and ultimately to IL-8 transcription. The full understanding of the S. aureus-induced biochemical processes in human conjunctival epithelium will bring new insight to the knowledge of the molecular mechanisms involved in conjunctiva bacterial infections and develop novel treatment aiming at phlogosis modulation.

Impact of diabetes on cognitive impairment and disability in elderly hospitalized patients with heart failure.

AIM: Heart failure (HF) and diabetes mellitus (DM) are each associated with cognitive impairment and disability. The aim of the present study was to evaluate the impact of DM on cognitive impairment and functional status in elderly hospitalized patients affected by HF. METHODS: A total of 79 elderly hospitalized patients with HF were enrolled in the present study. They underwent physical and instrumental examination, and geriatric multidimensional assessment including Mini-Mental State Examination (MMSE), Geriatric Depression Scale (GDS), activities of daily living (ADL) and instrumental activities of daily living (IADL). Differences between groups were established by t-test, Spearman's correlation coefficient was searched to examine the relation between variables. All results were considered significant if P was <0.05. RESULTS: HF and DM coexisted in 43 patients (54.4% of cases); when they occurred together patients showed, compared with non diabetic patients, a greater clinical severity of HF (44.2% were in New York Heart Association class IV vs 16.7%, P = 0.017), a lower MMSE score (20.4 ± 3.6 vs 23 ± 3.8, P = 0.004), and a lower number of preserved functions in ADL (3 ± 1.6 vs 4 ± 1.8, P = 0.008) and in IADL (3.2 ± 1.7 vs 4.6 ± 2.3, P = 0.003). The correlation between DM and cognitive impairment, and disability was confirmed by multivariate and univariate analysis. CONCLUSIONS: We confirm that DM is frequent in elderly hospitalized patients with HF, and we report that it has a negative impact on cognitive functions and functional status, worsening cognitive impairment, and disability observed in these patients. Comprehensive geriatric assessment is necessary for older adults with HF, especially when DM coexists.

Follicular variant of papillary thyroid carcinoma presenting as toxic nodule in an adolescent: coexistent polymorphism of the TSHR and Gsα genes.

BACKGROUND: Autonomously functioning, "hot", thyroid nodules are not common in children and adolescents. Such nodules are not considered alarming because they are assumed to be benign adenomas. Herein, we present a 15-year-old girl with a papillary thyroid carcinoma of 3.5 cm in diameter, which was functionally autonomous and scintigraphically hot. PATIENT FINDINGS: The patient, initially referred to our Endocrine Unit because of a thyroid nodule, returned 6 months later for symptoms of hyperthyroidism. Hyperthyroidism was confirmed biochemically. Radioactive iodine ((131)I) thyroid scintigraphy was consistent with an autonomous thyroid nodule. As per guidelines, the patient underwent surgery and a pathological examination revealed papillary carcinoma, follicular variant. The excised nodule was examined for activating mutations of the thyrotropin receptor (TSHR), Gsα (GNAS1), H-RAS, N-RAS, K-RAS, and BRAF genes by direct sequencing. No mutations were found. Nevertheless, two combined nonfunctioning mutations were detected: a single-nucleotide polymorphism (SNP) of the TSHR gene, in exon 7, at codon 187 (AAT→AAC, both encoding asparagine), and a SNP within exon 8 of the Gsα gene at codon 185 (ATC→ATT, both encoding isoleucine). Both SNPs were also identified in the germline DNA of the patient. The same SNPs were sought in the parents and brother of our patient. Her father was heterozygous for the TSHR SNP, her mother heterozygous for the Gsα SNP, and her brother was wild type. CONCLUSIONS: This case demonstrates that the presence of hyperfunctioning thyroid nodule(s) does not rule out cancer and warrants careful evaluation, especially in childhood and adolescence to overlook malignancy.

Electrocardiographic changes in centenarians: a study on 42 subjects and comparison with the literature.

BACKGROUND: Aging is associated with extensive and pervasive changes in cardiovascular structure and function, which may result in electrocardiographic alterations. The typical modifications seen in an electrocardiogram (ECG) in elders are: prolonged PR and QT intervals, QRS left-axis deviation and microvolt T wave. Several studies have included elderly people, but not long-living elderly and centenarians in particular who represent an increasing part of the population. OBJECTIVE: The aim of this study was to investigate the electrocardiographic findings in a population of centenarians and to compare the results with the few studies present in the literature. METHODS: We analyzed 42 healthy centenarians (12 males, 30 females; average age 101.43 ± 1.80 years) living in Messina, a municipality of Eastern Sicily, in Italy. They were clinically and functionally evaluated. All ECGs were analyzed by a single observer blind to clinical data. We assessed survival by phone recall. RESULTS: PR interval mean duration was 190 ± 3.3 ms, QRS 90 ± 1.4 ms, QTc interval mean duration was 370 ± 3.5 ms. Entirely normal ECG recordings were found in 7 centenarians (16.6%). The most frequently observed abnormalities included left-axis deviation and left anterior hemiblock in 16 centenarians (38.09%), left ventricular hypertrophy and aspecific ST-T wave abnormalities in 13 subjects (30.95%). We found no statistically significant differences between men and women. The mean age at death was 102.44 ± 2.45, and we did not find significant differences in age at death in long-living elderly in relation to different electrocardiographic findings. Comparing our results with two previous studies in the literature, the first conducted in Switzerland [Cornu: Rev Med Suisse Rom 1979;99:107-113] and the second in Nebraska [Lakkireddy et al.: Am J Cardiol 2003;92:1249-1251], we found a higher frequency of left ventricular hypertrophy that was compatible with the prevalence of hypertension in our centenarians (33.3%). Moreover, we did not find left bundle branch block, and the frequency of premature beats was remarkably less than that observed in the Swiss and US studies. CONCLUSIONS: Considering the increasing rate of centenarians, we believe that the results of the present study on electrocardiographic changes in centenarians may also be useful in clinical practice.

Healthy centenarians show high levels of circulating interleukin-22 (IL-22).

Aging is characterized by a progressive alteration of homeostatic mechanisms modulated by environmental and genetic factors. It is associated with a pro-inflammatory status. In centenarians, an increase of pro-inflammatory cytokine production balanced by anti-inflammatory immune response that would promote longevity is observed. Cytokine dysregulation is believed to play a key role in the proposed remodeling of the immune-inflammatory responses accompanying old age. IL-22 is a pro-inflammatory cytokine belonging to the IL-10 family and represents an important effector molecule of activated T helper (Th)-22, Th-1, and Th-17 cells. We recruited 17 healthy centenarians (4 males, 13 females, range 100-105 years). All ultralongeval subjects were living at home or in a nursing home. Sixteen healthy, sex-matched individuals (4 males, 12 females, range 60-95 years) were also recruited as controls. Centenarians displayed significantly higher circulating IL-22 levels compared to control population (45.7±66.9 pg/ml versus 11.1±6.5 pg/ml; p=0.031). It's well known that IL-22 is a pro-inflammatory cytokine produced by activated T lymphocytes and NK cells. IL-22 stimulates the production of acute phase reactants and promotes the antimicrobial defense. The results of the present study show, for the first time, that there is an increase of IL-22 in healthy centenarians. This pro-inflammatory condition probably is protective against infection, promoting the longevity of these subjects.

Association between oxidative stress and macromolecular damage in elderly patients with age-related macular degeneration.

BACKGROUND AND AIMS: The aim of the present study was to determine whether age and gender affect the imbalance between oxidant production and antioxidant levels in age-related macular degeneration (ARMD) patients. METHODS: Total superoxide dismutase (T-SOD), total glutathione peroxidase (T-GSHPx), and catalase (CAT) activities, as well as malondialdehyde (MDA), protein carbonyl (PC), 8-Hydroxy-29-deoxyguanosine (8-OHdG) and total oxidation status (TOS) levels, were measured in the following groups subdivided by age and gender: 156 early-ARMD patients; 80 wet-late ARMD patients; 72 dry-late ARMD patients; and 207 healthy controls. RESULTS: Among all study participants, women aged 50-54 had higher T-SOD and T-GSHPx activities and lower MDA, PC, TOS and 8-OHdG levels than age-matched men (p<0.05), whereas older women were not significantly different from agematched older men. Significantly increased oxidative damage was associated with ARMD patients >60 years of age in both sexes compared with controls (p<0.01 for 60-64 and 65-69-year-old ARMD subgroups; p<0.001 for 70-74 and 75-80-year-old ARMD subgroups). Multiple regression analysis demonstrates that age significantly affects antioxidant status and oxidative damage in ARMD patients compared with controls (controls, p<0.05; ARMD patients, p<0.001). A direct correlation with antioxidant enzyme activities and an inverse correlation with oxidative DNA, protein and lipid damage were also observed in premenopausal women (controls, p<0.05; ARMD patients, plt;0.001). CONCLUSIONS: Aging and postmenopausal status may be aggravating factors contributing to redox imbalance and oxidative damage in ARMD patients.

Proinflammatory gene expression at chronic periodontitis and peri-implantitis sites in patients with or without type 2 diabetes.

BACKGROUND: Diabetes and periodontal diseases are often associated. Both have highly inflammatory components, but the role played by distinct phlogistic mediators in their pathogenesis is not fully understood and remains controversial. The purpose of this study is to evaluate whether type 2 diabetes alters the expression of inflammatory mediators in sites with chronic periodontitis (CP) or peri-implantitis (P-IM). METHODS: The expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and -8, and monocyte chemotactic protein (MCP)-1 plus key CC chemokine receptors (CCR1 through 5) and CXC chemokine receptors (CXCR1 through 3) was quantified by real-time polymerase chain reaction (PCR) in gingival or peri-implant biopsies from 135 patients with well-controlled or poorly controlled diabetes and periodontal disease, 65 patients with periodontal disease but otherwise healthy, and 90 systematically and periodontally healthy subjects. Western blots were performed. RESULTS: Relative to controls, in patients without diabetes and patients with well-controlled diabetes, TNF-alpha, CCR5, and CXCR3 expression was exclusively higher in sites with P-IM (P <0.01), whereas IL-6 and -8 were overexpressed in sites with CP and, even more, in sites with P-IM (P <0.01). In patients with poor glycemic control, TNF-alpha, CCR5, and CXCR3 mRNAs were increased in sites with CP (P <0.01). A statistically significant higher IL-6 and -8 expression from patients without diabetes and patients with well-controlled diabetes was observed compared to patients with poorly controlled diabetes. Regardless of metabolic/glycemic status, MCP-1 and CCR2 and 4 were markedly higher in both of the oral pathologies examined (P <0.01). At the protein levels, Western blot experiments confirmed the real-time PCR results. CONCLUSIONS: These findings showed that: 1) in subjects without diabetes and patients with well-controlled diabetes, TNF-alpha, CCR5, and CXCR3 may constitute distinctive biomarkers of P-IM; 2) poor glycemic control abolished the differences between CP and P-IM regarding the expression of these mediators; and 3) type 2 diabetes affected the expression of TNF-alpha, IL-6 and -8, CCR5, and CXCR3.

Pseudomonas aeruginosa induces interleukin-8 (IL-8) gene expression in human conjunctiva through the recruitment of both RelA and CCAAT/enhancer-binding protein beta to the IL-8 promoter.

The purpose of this study was to identify the Pseudomonas aeruginosa-activated signaling pathway leading to interleukin (IL)-8 gene expression and protein synthesis by human conjunctival epithelium. IL-8 protein and mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription-PCR, respectively. Activation of MAPKs and NF-kappaB was analyzed by Western blotting using phosphospecific antibodies. We used transfection with wild-type or mutated IL-8 promoters and cotransfection with transcription factor overexpressing plasmids or small interfering RNAs. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) were performed for in vitro and in vivo protein-DNA binding studies, respectively. P. aeruginosa increased IL-8 expression at the transcriptional level by phosphorylating CCAAT/enhancer-binding protein beta (C/EBPbeta) via p38MAPK and activating NF-kappaB. The simultaneous involvement of RelA and C/EBPbeta and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. Re-ChIP experiments showed that RelA and C/EBPbeta act together at the IL-8 promoter level upon P. aeruginosa infection. Taken together, our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPbeta and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.

Regulation of interleukin-8 gene at a distinct site of its promoter by CCAAT enhancer-binding protein homologous protein in prostaglandin E2-treated human T cells.

For a long period of time, the transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) has been thought to inhibit transcriptional activity for its ability to interact with CCAAT enhancer-binding protein family factors, thus preventing their binding to DNA. We have previously shown that in human T lymphocytes the CHOP phosphorylation induced by prostaglandin E(2) (PGE(2))-increased interleukin-8 (IL-8) gene expression. Given the CHOP positive role in the regulation of transcription, here we have investigated the molecular mechanism(s) by which CHOP increases IL-8 gene activity under PGE(2) stimulus. Transfection experiments with mutants showed both that the CHOP transactivation domain is essential for IL-8 transcription and that the IL-8/activator protein 1 (AP-1) promoter mutated in NF-kappaB and NF-IL-6, but not in the AP-1 site, harbors essential CHOP-responsive elements. CHOP silencing confirmed its role in the IL-8 transcriptional regulation and protein production, whereas c-Jun small interfering RNA experiments showed that the PGE(2)-induced activation of IL-8 promoter is mainly c-Jun-independent. Moreover, PGE(2) induced CHOP-DNA complexes only when the entire IL-8/AP-1 promoter or the wild type sequences encompassing the AP-1 upstream region were employed. Mutations introduced in these sequences prevented the DNA-CHOP complex formation. The IL-8/AP-1 mutant promoter lacking the sequence immediately upstream the AP-1 site is PGE(2)-unresponsive. Finally, chromatin immunoprecipitation data confirmed in vivo that PGE(2) induces CHOP binding to the IL-8 promoter. Taken together, our results suggest that the increased expression of CHOP in response to PGE(2) exerts a positive transcriptional regulation of the IL-8 promoter mediated by direct binding to a novel consensus site.

Transcriptional regulation of IL-8 by Staphylococcus aureus in human conjunctival cells involves activation of AP-1.

PURPOSE: To identify signal transduction pathways involved in interleukin (IL)-8 expression by human conjunctival cells challenged with Staphylococcus aureus. METHODS: Conjunctival cells were cultured in the presence of live or heat-killed S. aureus. IL-8 protein and mRNA were determined by ELISA and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs) and NF-kappaB was analyzed by Western blot analysis with phosphospecific antibodies. Conjunctival cells were transfected with wild-type (wt) or mutated IL-8 promoters (IL-8-97, lacking the AP-1 site; IL-8-97 mutant C/EBP; IL-8-97 mutant NF-kappaB; IL-8/AP-1 double mutant for C/EBP and NF-kappaB) or c-Jun-NH(2)-terminal kinase (JNK)-responsive GAL-c-Jun. In further experiments, cells were cotransfected with wt IL-8 promoter and expression plasmids for p38MAPK-responsive C/EBP homologous protein (CHOP) or wt or dominant negative transactivation domain mutant (TAM-67) c-Jun. A protein-DNA binding study was performed by electrophoretic mobility shift assay (EMSA), to identify the transcription factors bound to the IL-8 promoter. RESULTS: S. aureus induced significant IL-8 expression and synthesis in human conjunctival epithelial cells by activating c-Jun phosphorylation and transactivation potential via JNK. The IL-8 promoter activation was NF-kappaB- and p38MAPK-independent. Transfection and EMSA experiments suggested that only AP-1 transcription factors were necessary for optimal IL-8 expression. CONCLUSIONS: Human conjunctival epithelial cells possess the ability to respond to Gram-positive S. aureus and to activate the innate immune response by the IL-8 gene expression. These results are the first to delineate the transcription factors involved in S. aureus-induced IL-8 release by conjunctival epithelium.

Concentration of circulating endothelial progenitor cells (EPC) in normal pregnancy and in pregnant women with diabetes and hypertension.

OBJECTIVE: The aim of the present study was to analyze the concentrations of endothelial precursor cells (EPCs) during the 3 trimesters of normal pregnancy and to compare the EPC counts in women with normal pregnancy, gestational diabetes, and gestational hypertension. STUDY DESIGN: The study was conducted on 21 pregnant women with single pregnancies (age range, 22 to 35 years). EPCs were quantified by flow cytometry. The subjects were divided into 3 groups, each consisting of 7 subjects: patients with gestational diabetes; patients with gestational hypertension; patients with normal pregnancy. RESULTS: A progressive increase was found in the concentrations of EPCs during pregnancy in healthy women. In the third trimester of pregnancy, the number of CD34+ cells was significantly lower in patients with gestational diabetes than in hypertensive patients and controls; no significant differences were found between the levels of circulating CD34+ cells in hypertensive patients and those in controls. There were no significant differences between the diabetic and hypertensive patients for the percentage of cells expressing CD133 and VEGFR2, whereas in both groups the percentage of CD133+/VEGFR2+ elements was significantly higher than in the healthy control subjects. CONCLUSION: Our findings confirm that EPCs isolated from the maternal circulation increase gradually throughout the gestational trimesters. These cells were derived from the endothelial cells lineage, as demonstrated by CD133+/VEGFR2+ cell assay. Moreover, the concentration of EPCs in pregnant women with gestational diabetes and hypertension differs from that in subjects with a normal pregnancy, CD34+ cells being reduced but CD133+/VEGFR2+ cell concentrations being increased. These results not only substantiate recent insights into the mechanisms regulating maternal vascular modifications during pregnancy but also throw light upon the activation of different patterns in the mobilization of endothelial progenitor cells during pathologic states in which endothelial disorders occur.