Barbara Marzocchi

Oxidative stress in preterm neonates at birth and on the seventh day of life.

Previous studies have demonstrated increased oxidative damage to proteins and increased lipid peroxidation products in the plasma of hypoxic newborns at birth. We tested the hypothesis that hypoxic preterm newborns are at increased risk for oxidative stress in the first week of life. Heparinized blood samples of 34 hypoxic and 15 control preterm newborns were obtained at birth from the umbilical vein immediately after delivery and from a peripheral vein on postnatal d 7. Plasma levels of hypoxanthine, total hydroperoxide (TH), and advanced oxidation protein products (AOPP) were measured in cord blood and blood drawn on d 7. Hypoxanthine, TH, and AOPP levels were significantly higher in cord and d 7 blood samples of hypoxic newborn than control infants. Statistically significant correlations were observed between AOPP and hypoxanthine and between AOPP and TH plasma levels on d 7. AOPP and TH plasma levels significantly increased from cord to d 7 blood in neonates without hypoxia. These findings show that the oxidative stress observed in cord blood of hypoxic preterm newborns is still higher than control infants on d 7. The significant increase in TH and AOPP levels in nonhypoxic preterm newborns at the end of the first postnatal week indicates that damage caused by free radicals also occurs in nonhypoxic babies with normal clinical course. In summary, TH and AOPP production is prolonged for several days after birth in hypoxic preterm babies. The risk of free radical damage is lower but still exists in preterm neonates with normal clinical course.

Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two‐dimensional electrophoresis map with patient sera

Western blots of two‐dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot‐cluster due to the chlamydia‐specific antigen outer membrane protein‐2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL‐like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface‐exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK‐like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N‐terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha‐subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF‐Tu, seven (41%) a putative stress‐induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl‐like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.

Effects of Lutein on Oxidative Stress in the Term Newborn: A Pilot Study

Background: Oxidative stress (OS) plays a crucial role in pathological conditions during the early neonatal period. The newborns are susceptible to oxidative damage due to high metabolic rate and low levels of antioxidant enzymes. Lutein has been found to have protective functions in adult humans as antioxidant. Aim: To evaluate the effects of lutein on OS in newborns. We tested the hypothesis that lutein would act both by increasing antioxidant capacity and inhibiting OS. Methods: This was a randomized, double-blind, placebo-controlled, single-center study. 20 healthy term newborns were assigned to receive lutein or placebo (lutein and control group, respectively) at 12 and 36 h after birth. Total hydroperoxides (TH), as marker of OS, and biological antioxidant potential (BAP), as marker of antioxidant power, were detected on cord blood and at 48 h of life in all babies. Results: TH significantly increased from birth to 48 h in the control group (p = 0.02), but not in the lutein group. In the lutein group, BAP significantly increased after 48 h (p = 0.02), showing a strengthening of antioxidant activity due to lutein. At 48 h of life, compared with those in the control group, neonates assigned to receive lutein had significantly lower TH levels (p = 0.04) and higher BAP levels (p = 0.028). Conclusions: Lutein administration in newborns increases the levels of BAP decreasing TH. The enhancement of antioxidant activity in plasma clearly results in protecting newborn from perinatal OS. These preliminary results, adding a new contribution in antioxidant strategies, strongly require to be confirmed by RCT.

Non-protein-bound iron detection in small samples of biological fluids and tissues.

Interest in the pro-oxidative nature of non-protein-bound-iron (NPBI) led to the development of an assay for its detection. The aim was to set up a reliable method of detecting NPBI in small samples of biological fluids and tissue. The method was based on preferential chelation of NPBI by a large excess of the low-affinity ligand nitrilotriacetic acid. To separate NPBI, a two-step filtration procedure was used. All glassware and plasticware were treated to minimize iron contamination. Measurements were performed in plasma, amniotic fluid, bronchoalveolar lavage, and brain tissues. The analytic system detected iron as ferric nitrate standard down to a concentration of 0.01 μM. The 1,2-dimethyl-3-hydroxy-4(1H)-pyridone-Fe(DHP-Fe) complex eluted with a retention time of about 2.6 min. The standard curve for the DHP-Fe complex was linear between 0.01 and 400 μM in water as well as in plasma, bronchoalveolar lavage, brain tissue, and amniotic fluid. The detection limit was 0.01 μM for all biological fluids and brain tissue. The data show that reliable measurements of NPBI are possible in studies on oxidative stress under experimental and clinical conditions. The possibility of investigating NPBI involvement in free-radical injury might be useful in all human diseases in which oxidative stress occur.

Nonprotein-bound iron and plasma protein oxidative stress at birth.

We previously reported plasma nonprotein-bound iron (NPBI) as a reliable early indicator of intrauterine oxidative stress (OS) and brain injury. We tested the hypothesis that albumin, an NPBI serum carrier, is the major target of NPBI-induced OS. Twenty-four babies were randomly selected from 384 newborns constituting the final cohort of a prospective study undertaken to evaluate the predictive role of NPBI in cord blood for neurodevelopmental outcome. Twelve were selected in the group with lowest NPBI levels (0–1.16 μM) and good neurodevelopmental outcome and 12 in the group with highest NPBI levels (≥15.2 μM) and poor neurodevelopmental outcome. Protein carbonyl groups were identified in cord blood samples by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blotting with anti-2,4-dinitrophenyl (DNP) antibodies. Two series of immunoreactive spots, corresponding to serum albumin and α-fetoprotein, were found only in the group with highest NPBI levels. We found an association between NPBI and carbonylated proteins in babies with highest NPBI levels. Since NPBI may produce hydroxyl radicals through the Fenton reaction, the major target of OS induced by NPBI is its carrier: albumin. Oxidation of albumin can be expected to decrease plasma antioxidant defenses and increase the likelihood of tissue damage due to OS in the newborns.

Perinatal outcome and placental histological characteristics: a single-center study

Objective: Placental pathology assists in characterizing the antenatal environment and may provide information about the baby’s subsequent development. We aim to assess whether histological patterns of placenta are associated with an increased risk of perinatal diseases and to evaluate how different patterns of placental dysfunction can affect the neurodevelopmental outcome. Methods: We analyzed the histopathological characteristics of 105 singleton placentas from infants born between 23 and 31 weeks of gestation and we assessed pair-wise correlations with perinatal diseases. Estimated relative risks were calculated from odds ratios. Results: Histological chorioamnionitis (CA group) was detected on 51 of 100 placentas tested. Lesions of uteroplacental circulation (abruption, infarction or thrombosis, perivillous fibrin deposition, syncytial knots; vasculopathy group) were detected on 29. 25 normal placentas served as controls. The incidence of bronchopulmonary dysplasia (BPD) and patent ductus arteriosus (PDA) was higher in CA than in control group. The risk of developing retinopathy of prematurity (ROP), intraventricular hemorrhage (IVH) and PDA was higher in CA than in vasculopathy group. Conclusions: At low gestational age CA, rather than placental lesions of vasculopathy, negatively impacts perinatal outcome. Clinical significance of histologic vasculopathy remains questionable. Other pathophysiological mechanisms than those associated with placental changes may occur following dysfunction of uteroplacental circulation.

Chondroptosis in Alkaptonuric Cartilage

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above‐mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. 230: 1148–1157, 2015.

Hypoxia-Induced Post-Translational Changes in Red Blood Cell Protein Map of Newborns

Tyrosine (Tyr) phosphorylation is implicated in the modification of several erythrocyte functions, such as metabolic pathways and membrane transport, as well as in signal transduction systems. Here we describe the map of Tyr-phosphorylated soluble proteins of newborn red blood cells (RBC) using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. We tested the hypothesis that a hypoxic environment and subsequent reoxygenation promote post-translational changes in the RBC protein map of newborns, in addition to desferrioxamine (DFO)-chelatable iron (DCI) release and methemoglobin (MetHb) formation. Umbilical cord blood RBC were incubated under hypoxic conditions for 16 h at 37°C, and subsequently for 8 h under aerobic conditions. Control erythrocytes were incubated under aerobic conditions at 37°C for the period of the experiment, i.e. for 24 h. Tyr-phosphorylation proteins were assessed using advanced high-resolution two-dimensional electrophoresis, 2-D immunoblot analysis with anti-phosphotyrosine (anti-pTyr) antibodies, and computer-aided electrophoretogram analysis. Higher DCI release and MetHb formation were observed in newborn RBC incubated under hypoxic conditions than in those incubated aerobically. Different immunoreactivity patterns with anti-pTyr antibodies were also observed between newborn RBC incubated under hypoxic conditions and controls. A hypoxic environment is a factor promoting DCI release, a well-known condition of oxidative stress. This is the first map of Tyr-phosphorylated soluble proteins of newborn RBC obtained using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. Our results suggest that hypoxia increases Tyr-phosphorylation of antioxidant proteins, protecting RBC against oxidative stress.

Evolutionary trends of neurofilament proteins in fish

1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.

Oxidative damage induced by herbicides is mediated by thiol oxidation and hydroperoxides production

Abstract Toxicological and environmental issues are associated with the extensive use of agricultural pesticides, although the knowledge of their toxic effects as commercial formulations is still far from being complete. This work investigated the impact of three herbicides as commercial formulations on the oxidative status of a wild type Saccharomyces cerevisiae strain. With yeast being a well-established model of eukaryotic cells, especially as far as regards the stress response, these results may be indicative of potential damages on higher eukaryotes. It was found that herbicide-mediated toxicity towards yeast cells could be the result of an increased production of hydroperoxides (like in the case of the herbicides Pointer and Silglif) or advanced oxidation protein products and lipid peroxidation (especially in the case of the herbicide Proper Energy). Through a redox-proteomic approach it was found also that, besides a common signature, each herbicide showed a specific pattern for protein thiols oxidation.