Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/microL (equivalent to 2465.8 molecules/microL) to 10-9 (equivalent to 2.46 molecules/microL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/microL and for the nested PCR was 2.46 molecules/microL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.     

Early use of Nasal-BiPAP in two infants with Congenital Central Hypoventilation syndrome

Aim: To reduce the problems caused by prolonged artificial ventilation in babies with Congenital Central Hypoventilation syndrome (CCHS). Methods: Two term infants with CCHS, weighing 4030 g and 3100 g, respectively, at the beginning of treatment and aged 53 and 31 d, respectively, were successfully ventilated with a Nasal Bilevel Positive Airway Pressure (N-BiPAP) device. Results: In the first patient the tcPO ₂ recordings (mean ± SD) during sleep were 46 ± 12 mmHg before using N-BiPAP and 58 ± 13 mmHg after using the device, while those for tcPCO ₂ were 75 ± 9 mmHg and 49 ± 11 mmHg, respectively. In the second patient tcPO ₂ during sleep was 42 ± 3 mmHg before, and 55 ± 5 after N-BiPAP, and for tcPCO ₂ the recordings were 119 ± 24 mmHg and 55 ± 6 mmHg, respectively, showing a significant improvement. One infant had persistent gastro-oesophageal reflux, and frontal skin abrasion caused by the face mask. Nevertheless, these complications did not necessitate the discontinuation of N-BiPAP ventilation, thus precluding prolonged use of intubation and tracheotomy.

Conclusion: In infants with CCHS, early use of non-invasive, positive-pressure ventilation with N-BiPAP, in association with careful monitoring, can decrease problems caused by prolonged intubation and tracheotomy.     

Pregnancy and autoimmunity: maternal treatment and maternal disease influence on pregnancy outcome

If a woman suffers from autoimmune disease (AD), several factors can affect pregnancy or neonatal outcome: repeated spontaneous pregnancy losses (frequently related to antiphospholipid antibodies (aPL)), neonatal lupus with complete congenital heart block (CHB) (linked to transplacental passage of IgG anti Ro/SS-A antibodies) and the disease activity itself that can affect the mother, the pregnancy and fetal outcome. If appropriately managed, the antiphospholipid syndrome (APS) is "one of the few tractable causes of pregnancy losses." A recent case control study, on babies from APS-mothers and healthy mothers, did not show any difference in the occurrence of neonatal complications. There are few data about the long-term outcome of babies born to patients with AD. We recently reported increased occurrence of learning disabilities in children born to aPL positive mothers with systemic lupus erythematosus (SLE). The modern management of pregnancy in patients with AD includes the treatment of disease flares, using drugs effective but safe for fetus. Corticosteroids and some immunosuppressive drugs can be used in pregnancy to control maternal disease. A prolonged fetal exposure to dexamethasone was reported to impair cerebral development, but we recently studied 6 children, born to patients treated with dexamathasone because of CHB, showing a normal intelligence quotient. The last 10-year experience shows that fetal exposure to antimalarial drugs should not be regarded as an important risk factor for gestational nor neonatal complications. However, information about long-term outcome of children exposed to immunosuppressive drugs "in utero" are still lacking and more efforts are needed in this research area.     

Efficacy of human recombinant erythropoietin plus IFN-alpha in patients affected by chronic hepatitis C.

Determination of serum iron levels in patients affected by chronic hepatitis C is considered fundamental for studying the response to interferon-alpha (IFN-alpha) treatment. IFN could induce anemia, which is promptly corrected by exogenous administration of recombinant human erythropoietin (rHuEPO). The aim of our study was to verify the possible beneficial effect of rHuEPO in patients affected by chronic hepatitis C and treated with IFN. Seventy consecutive patients (42 males and 28 females, mean age 46.4+/-5.2 years) affected by chronic hepatitis C were enrolled. In all patients, chronic hepatitis C was diagnosed on the basis of clinical and biological findings (alanine aminotransferase [ALT] serum levels at least 2-fold higher than normal values for at least 12 months and the presence of anti-HCV antibodies). All patients were negative for hepatitis B virus (HBV) infection, hepatitis D virus (HDV infection, and HIV infection. Statistical analysis was carried out using the Wilcoxon nonparametric sum rank test, the Spearman correlation rank test, and the Friedman ANOVA and Kendall coefficient of concordance. At the end of the treatment, our study series showed significant differences in serum levels of AST (p < 0.001), iron (p < 0.001), and ferritin (p < 0.001). At the end of the follow-up period, significant differences were seen in ALT, aspartate (AST), and iron ferritin and transferrin levels. All differences favored patients who received IFN-alpha and rHuEPO. We think that the depletion of circulating iron may improve the immune response impaired by iron accumulation in the liver. Our study confirms the important role played by iron in the response to IFN treatment, suggesting that the use of rHuEPO induces a better response to IFN in patients with chronic hepatitis C by activation of erythropoiesis.     

A uterovaginal septum and imperforate hymen with a double pyocolpos

The presence of both a uterovaginal septum and imperforate hymen is described in a young patient presenting with ongoing chronic pelvic pain and a double pyocolpos. Ultrasound and magnetic resonance imaging scans were performed. The patient underwent laparoscopic adesiolysis, hymenotomy with drainage of 200 mL of pus, and excision of a complete longitudinal vaginal septum. Over the past 5 years of regular follow-up examinations, the patient has always reported regular menstrual cycles and an absence of pelvic pain.     

Modifications of Phleum pratense grass pollen allergens following artificial exposure to gaseous air pollutants (O(3), NO(2), SO(2))

BACKGROUND: Air pollution is frequently proposed as a potential cause of the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens. METHODS: Timothy grass pollen was exposed to ozone (O(3)), nitrogen dioxide (NO(2)) and sulphur dioxide (SO(2)) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. RESULTS: For O(3)-treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, Phl p 4, Phl p 5 and Phl p 6 and an IgE recognition decrease in Phl p 1, Phl p 2, Phl p 6 and Phl p 13. NO(2) exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO(2) treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO(2)/O(3) or NO(2)/SO(2) showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O(3) acidification was also observed with the NO(2)/O(3) mix. CONCLUSION: Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or post-translational modifications affecting allergen recognition by IgE.     

Involvement of PKCα–MAPK/ERK-phospholipase A2 pathway in the Escherichia coli invasion of brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood–brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA₂s siRNA. cPLA₂ and iPLA₂ enzyme activities and cPLA₂ phosphorylation were stimulated after E. coli incubation and were attenuated by PLA₂, PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.     

New mutations in the GLA gene in Brazilian families with Fabry disease

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the alpha-galactosidase A (GLA) gene. Evaluating the enzymatic activity in male individuals usually performs the diagnosis of the disease, but in female carriers the diagnosis based only on enzyme assays is often inconclusive. In this work, we analyzed 568 individuals from 102 families with suspect of FD. Overall, 51 families presented 38 alterations in the GLA gene, among which 19 were not previously reported in literature. The alterations included 17 missense mutations, 7 nonsense mutations, 7 deletions, 6 insertions and 1 in the splice site. Six alterations (R112C, R118C, R220X, R227X, R342Q and R356W) occurred at CpG dinucleotides. Five mutations not previously described in the literature (A156D, K237X, A292V, I317S, c.1177_1178insG) were correlated with low GLA enzyme activity and with prediction of molecular damages. From the 13 deletions and insertions, 7 occurred in exons 6 or 7 (54%) and 11 led to the formation of a stop codon. The present study highlights the detection of new genomic alterations in the GLA gene in the Brazilian population, facilitating the selection of patients for recombinant enzyme-replacement trials and offering the possibility to perform prenatal diagnosis.     

Ring 2 chromosome: ten-year follow-up report.

C?té et al. [1981: Ann Genet 24:231-235] suggested that ring chromosomes without a preceding deletion share a common pattern of phenotypic anomalies, independent of what chromosome is involved. The phenotype of such a "general ring syndrome" consists of growth failure without malformations, few or no minor anomalies, and mild-to-moderate mental retardation. We report on a patient with a ring 2 chromosome with features suggestive of Silver-Russell syndrome at birth and striking postnatal growth retardation with minor intellectual involvement supporting C?té's suggestion. This would be the ninth case of ring 2 chromosome published; the patient is the longest reported survivor, with a 10-year follow-up.     

Prostasomes inhibit the NADPH oxidase activity of human neutrophils

Prostasomes are particular lipid vesicles secreted by the prostate in human semen and involved in several physiological functions such as the improvement of sperm motility or immunomodulation. We have previously shown that they reduced the overall reactive oxygen species (ROS) production of seminal polymorphonuclear neutrophils (PMN). The present study was conducted to define the mechanism by which prostasomes inhibit the ROS production of blood and seminal PMN. The luminol chemiluminescence measuring total ROS production of blood PMN stimulated by either a phorbol ester (PMA) or a chemoattractant peptide, formyl-Met-Leu-Phe (fMLP) was significantly inhibited by prostasomes. The NADPH oxidase activity of the PMN was measured by 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-a]pyrazin-3-one (MCLA) chemiluminescence. Prostasomes inhibited the NADPH oxidase activity of blood or seminal PMN and increased the lag-phase of the enzyme after PMA stimulation. Prostasomes also inhibited significantly the NADPH oxidase activity of fMLP stimulated blood PMN, but the inhibition was not significant for seminal PMN. The lipid composition of blood PMN was analysed and compared to the lipid composition of prostasomes. This showed that prostasomes had a high cholesterol:phospholipid molar ratio and a high proportion of sphingomyelin. Together with the fact that prostasomes can rigidify the plasma membrane of blood PMN, these results led us to postulate that prostasomes inhibit the NADPH oxidase activity of PMN by lipid transfer from the prostasomes to the plasma membrane of the PMN.