Azithromycin therapy for prevention of chronic lung disease of prematurity (AZTEC): a multicentre, double-blind, randomised, placebo-controlled trial.

BACKGROUND: Systematic reviews have reported conflicting evidence on whether macrolide antibiotics reduce rates of chronic lung disease of prematurity (CLD) in at-risk preterm infants born at less than 30 weeks' gestation, including in those colonised with pulmonary Ureaplasma spp. Since an adequately powered trial has been lacking, we aimed to assess if the macrolide azithromycin improved survival without the development of physiologically defined moderate or severe CLD in preterm infants. METHODS: AZTEC was a multicentre, double-blind, randomised, placebo-controlled trial conducted in 28 tertiary neonatal intensive care units in the UK. Infants were eligible if they were born at less than 30 weeks' gestation and had received at least 2 h of either non-invasive (continuous positive airway pressure or humidified high flow nasal cannula therapy) or invasive respiratory support (via endotracheal tube) within 72 h of birth. Eligible infants were randomly allocated in a 1:1 ratio using random permuted blocks of four to receive either intravenous azithromycin at 20 mg/kg per day for 3 days followed by 10 mg/kg for 7 days, or to placebo. Allocation was stratified by centre and gestational age at birth (<28 weeks vs ≥28 weeks). Azithromycin and placebo vials were encased in tamper-evident custom cardboard cartons to ensure masking for clinicians, parents, and the research team. The primary outcome was survival without development of physiologically defined moderate or severe CLD at 36 weeks' postmenstrual age. Outcomes and safety were analysed on an intention-to-treat basis (all randomly allocated infants, regardless of any post-randomisation events). The study was registered with ISRCRN (11650227) and is closed. FINDINGS: Infants were recruited between Oct 9, 2019, and March 22, 2022. 799 (53·1%) of 1505 eligible infants underwent random allocation; three infants were withdrawn, including consent to use their data, leaving 796 infants for analysis. Survival without moderate or severe CLD occurred in 166 (42%) of 394 infants in the intervention group and 179 (45%) of 402 in the placebo group (three-level adjusted OR [aOR] 0·84, 95% CI 0·55-1·29, p=0·43). Pulmonary Ureaplasma spp colonisation did not influence treatment effect. Overall, seven serious adverse events were reported for the azithromycin group (five graded as severe, two as moderate), and six serious adverse events were reported in the placebo group (two severe, two moderate, and two mild), as assessed by the local principal investigators. INTERPRETATION: Since prophylactic use of azithromycin did not improve survival without development of physiologically-defined CLD, regardless of Ureaplasma spp colonisation, it cannot be recommended in clinical practice. FUNDING: UK National Institute for Health and Care Research.

APC transcription studies and molecular diagnosis of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is characterised by the development of hundreds to thousands of colorectal adenomas and results from inherited or somatic mosaic variants in the APC gene. Index patients with suspected FAP are usually investigated by APC coding region sequence and dosage analysis in a clinical diagnostic setting. The identification of an APC variant which is predicted to alter protein function enables predictive genetic testing to guide the management of family members. This report describes a 4-generation family with a phenotype consistent with FAP, but in which an APC variant had not been identified, despite testing. To explore this further, quantitative PCR (qPCR) was employed to assess APC transcription, demonstrating reduced levels of APC RNA. Next generation sequencing (NGS) identified the APC 5'UTR/ Exon 1 variant, c.-190 G>A, that had been reported previously in an another FAP family with APC allelic imbalance. Quantitative RNA studies and DNA sequencing of the APC promoters/ Exon 1 may be useful diagnostically for patients with suspected FAP when coding region variants cannot be identified.

Severity of left ventricular dysfunction in heart failure patients affects the degree of serum-induced cardiomyocyte apoptosis. Importance of inflammatory response and metabolism.

BACKGROUND/OBJECTIVES: In heart failure pro-inflammatory cytokines contribute to cardiomyocytes loss by apoptosis and play a role in the remodelling of the extracellular matrix (ECM). Myocardial injury recruits endothelial progenitor cells (EPCs) to the site of damage and stimulates their differentiation, contributing to myocardial tissue repair. We investigated if the severity of left ventricular dysfunction in heart failure patients (HF) may influence the ability of serum to induce cardiomyocytes death and whether this effect is affected by inflammation and intracellular oxidative stress pathways. METHODS: Adult murine cardiomyocytes HL-5 were incubated with 2% human serum from patients with heart failure (NYHA classes I to IV). Apoptosis was analysed by two different methods. TNF-α, IL-1ß, IL-6, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured in sera from patients. RESULTS: Cytokine levels were higher in sera from moderate-severe CHF compared to that of patients with mild CHF. Levels of CD117(+) (c-Kit(+)) cells and EPCs were significantly lower in blood from moderate-severe HF patients. Serum from HF patients induced a significantly higher ROS production involving p38 MAPK signalling and apoptosis in cardiomyocytes. NAC treatment prevented serum-induced oxidative effects. The increase of AMPK phosphorylation showed an involvement of FFA ß-oxidation during apoptotic stress. CONCLUSIONS: All these alterations could be used as predictive factors of worsening in heart failure and culture of cardiomyocytes could be employed to test pharmacological effects.

Effects of a short-term exercise training on serum factors involved in ventricular remodelling in chronic heart failure patients.

OBJECTIVES: We studied the effect of a short-term (3 weeks) exercise training program on the number of circulating CD34/KDR(+) endothelial progenitor cells (EPCs) and on serum levels of matrix metalloproteinases (MMPs) in chronic heart failure (CHF) patients as well as on serum capacity to foster colony forming units-endothelial cells (CFU-ECs) in vitro. METHODS: Effectiveness of training was assessed by the 6-minute walking test (6MWT). Peripheral blood and serum were obtained from fourteen patients with CHF due to coronary artery disease before and after an inpatient aerobic exercise training program. At admission and at discharge we analysed circulating EPC number and serum levels of MMPs, TIMP-1 and TNF-α. The number and function of CFU-EC colonies were evaluated in cultures performed with serum obtained before and after training. RESULTS: After training, distance walked at 6MWT and number of circulating CD34/KDR(+) cells increased (from 154 ± 27 to 233 ± 48 m; P<0.0001 and from 5 ± 3 to 9 ± 6 cells/ml P<0.05, respectively). Conversely, serum concentrations of MMP-1 and TIMP-1 decreased significantly (from 11.4 ± 2.4 to 6.3 ± 1.1 ng/ml, and from 320.4 ± 41.2 to 167.2 ± 12.6 ng/ml, respectively, both P<0.01), while MMP2/TIMP-1 and MMP-9/TIMP-1 ratios increased. Interestingly, we found increased CFU-EC proliferation in cultures performed with serum obtained after training. CONCLUSIONS: Considering that both EPCs and MMPs might play a role in vascular remodeling, the increased number of EPCs and MMP activities observed in this study, suggest that the selected short-term exercise training could be a potential therapeutic strategy to rescue cardiac function in CHF patients.

Sildenafil reduces insulin-resistance in human endothelial cells.

BACKGROUND: The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Human umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine. CONCLUSIONS/SIGNIFICANCE: These data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients.

Salt intake induces epithelial-to-mesenchymal transition of the peritoneal membrane in rats.

BACKGROUND: Dietary salt intake has been linked to hypertension and cardiovascular disease through volume-mediated effects. Accumulating evidence points to direct negative influence of salt intake independent of volume overload, such as cardiac and renal fibrosis, mediated through transforming growth factor beta (TGF-beta). Epithelial-to-mesenchymal transition (EMT) has been implicated as a key process in chronic fibrotic diseases, such as chronic kidney disease or heart failure. The potential role of dietary salt intake on cell transdifferentiation has never been investigated. This study analysed the effect of dietary salt intake on EMT and fibrosis in the peritoneal membrane (PM) in a rat model. METHODS: Twenty-eight Wistar rats were randomized to a normal salt (NS) or a high salt (HS) intake. NS and HS rats had free access to tap water or NaCl 2% as drinking water, respectively. After 2 weeks, samples of peritoneum were taken, and TGF-beta(1), Interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) mRNA expression were quantified with qRT-PCR. Fibrosis and submesothelial PM thickness were scored. EMT was evaluated using fluorescence staining with cytokeratin and alpha smooth muscle actin (alpha-SMA). RESULTS: Dietary salt intake caused peritoneal fibrosis and thickening of the submesothelial layer and induced EMT as identified by colocalization of cytokeratin and alpha-SMA in cells present in the submesothelial layer. Peritoneal TGF-beta(1) and IL-6 mRNA expression were upregulated in the HS group. CONCLUSION: High dietary salt intake induces EMT and peritoneal fibrosis, a process coinciding with upregulation of TGF-beta1.

Peripheral blood mononuclear cells from mild cognitive impairment patients show deregulation of Bax and Sod1 mRNAs.

Elevated oxidative stress-induced apoptosis has been found in peripheral cells from patients with Alzheimer's disease (AD). Furthermore, treatment of lymphocytes from AD patients, with Abeta(1-42) and H(2)O(2) results in enhanced apoptosis. Mild cognitive impairment (MCI), a clinical condition between normal aging and AD, shares with AD a similar pattern of peripheral markers of oxidative stress. In this study we investigated spontaneous and H(2)O(2)-induced oxidative stress and apoptosis levels in peripheral blood mononuclear cells (PBMCs) from MCI and AD patients, as well as from Parkinson's disease (PD) patients without cognitive impairment or age-matched healthy control. Sod1 mRNA levels were studied to analyse the anti-oxidative pathway, while Bax and Bcl-2 mRNAs levels and PARP protein cleavage were monitored to study apoptosis. We found that the expression of Sod1 and Bax mRNAs was statistically higher in both MCI and AD patients compared to controls or PD subjects. Since Bcl-2 mRNA level was not different among groups, the Bax/Bcl-2 ratio was statistically higher in AD and MCI patients. PARP cleavage was also enhanced in PBMCs from MCI and AD individuals and this finding was associated with a higher level of spontaneous apoptosis. Interestingly, exposure to H(2)O(2) induced a significant decrease of Bcl-2 mRNA transcript, while Sod1 and Bax mRNAs levels were unchanged in PBMCs derived from MCI and AD patients. In conclusion, our results show that Bax and Sod1 mRNA levels are altered in PBMCs from both MCI and AD patients and indicate these changes as potential biomarkers in the early diagnosis of AD.

Gonosomal mosaicism for a nonsense mutation (R1947X) in the NF1 gene in segmental neurofibromatosis type 1.

Segmental neurofibromatosis type 1 (SNF1), characterized by the regionally limited distribution of neurofibromatosis type 1 (NF1) features, has been attributed to mosaicism for an NF1 gene mutation. The occurrence of classical NF1 in the offspring of a parent with SNF1 suggests that cutaneous mosaicism may be accompanied by gonadal mosaicism. We studied a girl with generalized NF1, and her mother who has SNF1. A recurrent nonsense mutation in exon 31 (R1947X) of the NF1 gene was identified in the lymphocyte DNA of the affected child by denaturing high-performance liquid chromatography and PCR/direct sequencing. DNA sequence analysis failed, however, to identify the R1947X mutation in peripheral lymphocytes, and in keratinocytes and fibroblasts cultured from affected and unaffected skin in the mother. DNA fragments containing exon 31 were then cloned from each cell line and these clones were screened using allele-specific PCR. The R1947X mutation was identified in 29 of 146 clones derived from keratinocytes from the affected region and in 12 of 136 clones derived from fibroblasts from the affected region, but in no clones derived from clinically unaffected tissues. These findings confirm that gonosomal mosaicism can occur in SNF1, with consequent important implications for genetic counselling.

Unusual features in a patient with neurofibromatosis type 1: multiple subcutaneous lipomas, a juvenile polyp in ascending colon, congenital intrahepatic portosystemic venous shunt, and horseshoe kidney.

We report a case that draws attention to a hitherto undescribed association of neurofibromatosis type 1 (NF1) with juvenile polyp, congenital intrahepatic portosystemic venous shunt, multiple subcutaneous lipomas, and horseshoe kidney. Our patient has fulfilled the National Institutes of Health consensus conference criteria for NF1 by having neurofibromas, axillary freckling, Lisch nodules, and café-au-lait spots. There is no family history of NF1 and his 7-year-old son has no stigmata of NF1. On the other hand, the patient's family had a presumably dominant inheritance of horseshoe kidney: the father, proband, sister, and son of the other sister had a horseshoe kidney. The patient was investigated for mutations in the NF1 gene and PTEN, but no germline mutations were detected. The differential diagnosis for such a collection of hamartomatous, cutaneous, and vascular disorders includes the Proteus, Bannayan-Riley-Ruvalcaba, and Cowden syndromes. None of these diagnoses was convincingly confirmed in this patient.

Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors.

One of the main features of neurofibromatosis type 1 (NF1) is benign neurofibromas, 10-20% of which become transformed into malignant peripheral nerve sheath tumors (MPNSTs). The molecular basis of NF1 tumorigenesis is, however, still unclear. Ninety-one tumors from 31 NF1 patients were screened for gross changes in the NF1 gene using microsatellite/restriction fragment length polymorphism (RFLP) markers; loss of heterozygosity (LOH) was found in 17 out of 91 (19%) tumors (including two out of seven MPNSTs). Denaturing high performance liquid chromatography (DHPLC) was then used to screen 43 LOH-negative and 10 LOH-positive tumors for NF1 microlesions at both RNA and DNA levels. Thirteen germline and 12 somatic mutations were identified, of which three germline (IVS7-2A>G, 3731delT, 6117delG) and eight somatic (1888delG, 4374-4375delCC, R2129S, 2088delG, 2341del18, IVS27b-5C>T, 4083insT, Q519P) were novel. A mosaic mutation (R2429X) was also identified in a neurofibroma by DHPLC analysis and cloning/sequencing. The observed somatic and germline mutational spectra were similar in terms of mutation type, relative frequency of occurrence, and putative underlying mechanisms of mutagenesis. Tumors lacking mutations were screened for NF1 gene promoter hypermethylation but none were found. Microsatellite instability (MSI) analysis revealed MSI in five out of 11 MPNSTs as compared to none out of 70 neurofibromas (p=1.8 x 10(-5)). The screening of seven MPNSTs for subtle mutations in the CDKN2A and TP53 genes proved negative, although the screening of 11 MPNSTs detected LOH involving either the TP53 or the CDKN2A gene in a total of four tumors. These findings are consistent with the view that NF1 tumorigenesis is a complex multistep process involving a variety of different types of genetic defect at multiple loci.

A large deletion (1.5 Mb) encompassing the neurofibromatosis type 1 (NF1) gene in a patient with sporadic NF1 associated with dysmorphism, mental retardation, and unusual ocular and skeletal features.

A 20 year old male patient with sporadic neurofibromatosis type 1 (NF1) is described with a large deletion (1.5 Mb) involving the NF1 gene, dysmorphism, mental retardation, and unusual ocular and skeletal features. Several NF1 patients with a large NF1 deletion and associated dysmorphism, and a large number of neurofibromas for their age have been described. This study indicates that such large deletions can also involve flanking loci which affect ocular and skeletal development.

Three different pathological lesions in the NF1 gene originating de novo in a family with neurofibromatosis type 1.

Three members of a Portuguese family, who exhibited clinical evidence of neurofibromatosis type 1 (NF1), were found to possess different heritable and pathological mutations in their NF1 genes: a 1.5-Mb deletion spanning the entire NF1 gene, a truncating CGA-->TGA transition in exon 22 (R1241X), and a frameshift mutation in exon 29 (5406insT). All three lesions occurred de novo and are likely to have been generated by different mutational mechanisms. At least two of the mutations occurred on different chromosomal backgrounds. The probability of finding three non-identical NF1 gene lesions arising de novo in a family with NF1 is very remote, too low to be readily accepted as mere coincidence. A number of possible explanations for this unique finding were therefore explored, but none were found to be wholly convincing. This report nevertheless serves as a reminder that it is unwise, even in the case of an autosomal dominant condition, to extrapolate from the detection of a single mutation in a specific individual to assuming an identical molecular genetic aetiology in other clinically affected members of the same family.

Glial fibrillary acidic protein immunoreactive astrocytes in developing rat hippocampus.

The developmental pattern of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes was investigated in the hippocampus (subfields CA1, CA3 and CA4) and in the dentate gyrus of male and female rats aged 11, 16, 30, 90 and 150 days by immunohistochemistry associated with image analysis. Analysis was centred on stratum radiatum, a hippocampal area rich in GFAP-immunoreactive astrocytes. The volume of different portions of hippocampus, the number and the size of astrocytes, the intensity of cell body GFAP immunostaining as well as the extension of astrocyte were assessed. A maturation pattern consisting in higher cellular expression of GFAP, an increase in overall cell size and expanding arborisation from the 11th to the 30th postnatal day, followed by stabilisation of these parameters until the 90th day of life, and a subsequent decrease in the oldest age group studied was found. A sex-related different temporal pattern of astrocytes maturation in size and GFAP content was observed in the CA1 subfield only. The increase of GFAP content during pre-weaning ages was less pronounced in females than in males as well as the decrease between the 90th and the 150th day of age. Moreover, the size of astrocytes was larger in females than in males at the 11th and 150th days of life. These findings suggest that hippocampal astrocytes undergo rapid maturation in the 1st month of postnatal life, followed by a slow consolidation of this process until the 3rd month of life. At 5 months of age, there are still dynamic changes in the mature astrocytes, which become slender and thinner probably as a response to the increased volume of hippocampus noticeable at this age.

The hippocampus in spontaneously hypertensive rats: an animal model of vascular dementia?

Hypertension is a main risk factor for cerebrovascular disease, including vascular dementia. The present study was designed to evaluate if hypertension-dependent changes of the hippocampus of spontaneously hypertensive rats (SHR) of different ages were related with those occurring in vascular dementia. The hippocampus was chosen as the brain area involved in learning and memory. Systolic pressure was slightly increased in 2-month-old SHR in comparison with age-matched normotensive Wistar-Kyoto (WKY) rats and augmented progressively with age in SHR. No microanatomical changes were observed in the hippocampus of SHR of 2 months in comparison with age-matched WKY rats. A limited decrease of white matter volume was observed in 4-month-old SHR. In SHR of 6 months, a reduction of grey matter volume both in the CA1 subfield and in the dentate gyrus occurred. Evaluation of phosphorylated 200-kDa neurofilament immunoreactivity revealed a decreased immune reaction area in the CA1 subfield of 6-month-old SHR compared to age-matched WKY rats and no changes in the expression and localization of the dendritic marker microtubule associated protein (MAP)-2. In 6-month-old SHR, an increase of glial fibrillary acidic protein (GFAP)-expression was found by Western blot analysis. Immunohistochemistry revealed an increase in number (hyperplasia), but not in size of astrocytes. These findings indicate the occurrence of cytoskeletal breakdown and astroglial changes primarily in the CA1 subfield of the hippocampus of SHR of 6 months. The occurrence in the hippocampus of SHR of regressive changes and astroglial reaction similar to those occurring in neurodegenerative disorders with cognitive impairment suggests that they represent an animal model of vascular dementia.