Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations.

Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spectroscopy was used to identify dehydration and storage induced conformational changes in isolated DNA and sperm chromatin. Furthermore, hydrogen bonding in the preservation solutions associated with storage stability were investigated. Reactive oxygen species and DNA damage in dried sperm samples were found to accumulate with increasing storage temperature and storage duration. Non-reducing disaccharides (i.e., trehalose, sucrose) and albumin counteracted oxidative stress and preserved sperm chromatin during dried storage, whereas glucose increased DNA damage during storage. When sperm was dried in the presence of trehalose and albumin, no spectral changes were detected during storage at refrigeration temperatures, whereas under accelerated aging conditions, i.e., storage at 37 °C, spectral changes were detected indicating alterations in sperm chromatin structure.

Spectroscopic assessment of oxidative damage in biomolecules and tissues.

Oxidative damage is one of the main causes of cryopreservation injury compromising the use of cryopreserved biospecimens. The aim of this study was to evaluate the use of Fourier transform infrared spectroscopy (FTIR) as a non-invasive method to assess changes in biomolecular composition and structure, associated with oxidative stress in isolated biomolecules, acellular heart valve tissues, and ovarian cortex tissues. FTIR spectra of these specimens subjected to various treatments (H2O2- and Fenton-treatment or elevated temperatures) were vector normalized and selected spectral regions were analyzed by principal component analysis (PCA). Control and damaged biomolecules can easily be separated using PCA score plots. Acellular heart valve tissues that were subjected to different levels of oxidative damage formed separate cluster in PCA score plots. In hydrated ovarian tissue, large variation of the principal components was observed. Drying the ovarian tissues samples resulted in improved cluster separation of treatment groups. However, early signs of oxidative damage under mild stress conditions could not be detected by PCA of FTIR spectra. For the ovarian tissue samples, the standardly used nitro blue tetrazolium chloride (NBT) assay was used to monitor the amount of formazan production, reflecting reactive oxygen species (ROS) production at various temperatures. At 37 °C, formazan staining rapidly increased during the first 30 min, and then slowly reached a saturation level, but also at lower temperatures (i.e. 4 °C) formazan production was observed. In summary, we conclude that ATR-FTIR combined with PCA can be used to study oxidative damage in biomolecules as well as in tissues. In tissues, however, sample heterogeneity makes it difficult to detect early signs of oxidative damage.

Spectral fingerprinting to evaluate effects of storage conditions on biomolecular structure of filter-dried saliva samples and recovered DNA.

Saliva has been widely recognized as a non-invasive, painless and easy-to-collect bodily fluid, which contains biomarkers that can be used for diagnosis of both oral and systemic diseases. Under ambient conditions, salivary biomarkers are subject to degradation. Therefore, in order to minimize degradation during transport and storage, saliva specimens need to be stabilized. The aim of this study was to investigate the feasibility of preserving saliva samples by drying to provide a shelf-stable source of DNA. Human saliva was dried on filters under ambient conditions using sucrose as lyoprotective agent. Samples were stored under different conditions, i.e. varying relative humidity (RH) and temperature. In addition to assessment of different cell types in saliva and their DNA contents, Fourier transform infrared spectroscopy (FTIR) was used to evaluate the effects of storage on biomolecular structure characteristics of saliva. FTIR analysis showed that saliva dried without a lyoprotectant exhibits a higher content of extended ß-sheet protein secondary structures compared to samples that were dried with sucrose. In order to evaluate differences in characteristic bands arising from the DNA backbone among differently stored samples, principal component analysis (PCA) was performed, allowing a clear discrimination between groups with/without sucrose as well as storage durations and conditions. Our results indicated that saliva dried on filters in the presence of sucrose exhibits higher biomolecular stability during storage.

Increasing storage stability of freeze-dried plasma using trehalose.

Preservation of blood plasma in the dried state would facilitate long-term storage and transport at ambient temperatures, without the need of to use liquid nitrogen tanks or freezers. The aim of this study was to investigate the feasibility of dry preservation of human plasma, using sugars as lyoprotectants, and evaluate macromolecular stability of plasma components during storage. Blood plasma from healthy donors was freeze dried using 0-10% glucose, sucrose, or trehalose, and stored at various temperatures. Differential scanning calorimetry was used to measure the glass transition temperatures of freeze-dried samples. Protein aggregation, the overall protein secondary structure, and oxidative damage were studied under different storage conditions. Differential scanning calorimetry measurements showed that plasma freeze-dried with glucose, sucrose and trehalose have glass transition temperatures of respectively 72±3.4°C, 46±11°C, 15±2.4°C. It was found that sugars diminish freeze-drying induced protein aggregation in a dose-dependent manner, and that a 10% (w/v) sugar concentration almost entirely prevents protein aggregation. Protein aggregation after rehydration coincided with relatively high contents of ß-sheet structures in the dried state. Trehalose reduced the rate of protein aggregation during storage at elevated temperatures, and plasma that is freeze- dried plasma with trehalose showed a reduced accumulation of reactive oxygen species and protein oxidation products during storage. In conclusion, freeze-drying plasma with trehalose provides an attractive alternative to traditional cryogenic preservation.

[Echocardiographic evaluation of anti-tumor necrosis factor-alpha therapy with infliximab in patients without cardiac pathologies]. / Effetti del trattamento anti-Tumor Necrosis Factor-alpha con infliximab in pazienti non cardiopatici: valutazione ecocardiografica.

In the course of heart failure, plasmatic levels of Tumor Necrosis Factor-alpha (TNF-alpha) are high and are related to prognosis and mortality. Infliximab, a recombinant chimeric antibody anti-TNF-alpha, was used in heart failure with disappointing results, similar to those obtained with other biological drugs.The aim of this study was the echocardiographic evaluation of infliximab infusion in nine patients without cardiac pathologies. The findings demonstrate a reduction of sistolic function and a modification of diastolic function after infliximab infusion in patients without cardiopathy. This study confirms the protective role played by TNFalpha on the myocardium, as suggested by previous experimental studies.

Cardiac involvement in Crohn's disease: echocardiographic study.

BACKGROUND: Crohn's Disease (CD) commonly presents extra-intestinal manifestations, but cardiac involvement is considered rare. The aim of the present study was to assess cardiac involvement in CD and its possible correlation with activity, duration, localization and therapy. PATIENTS AND METHODS: A group of 68 patients with CD and a control group of 60 healthy subjects were subjected to a transthoracic echocardiogram with Doppler study. RESULTS: The study found overall morphologic alterations in 47/68 CD patients (69.11%) versus 12/60 controls (20.0%; P < 0.01); mitral valve prolapse in 20/68 CD patients (29.4%) versus 4/60 controls (6.6%; P < 0.01); and pericardial effusion in 13/68 CD patients (19.1%)versus 1/60 controls (1.6%; P < 0.01). The following findings were frequent, but without statistical significance: mitral insufficiency, 9/68 CD (13.2%) versus 3/60 controls (5.0%); tricuspidalic insufficiency, 8/68 CD (11.7%) versus 3/60 controls (5%); aortic insufficiency, 3/68 CD (4.4%) versus none in the control group; and decreased left ventricle ejection fraction, 5/68 CD (7.3%) versus none in the control group. Pericardial effusion was found to be related to CD activity (r = 0.375; P = 0.002) as well as decreased ejection fraction (r = 0.358; P = 0.003). No correlation with age, sex, duration, therapy or localization of disease was found. CONCLUSIONS: These findings suggest that CD frequently determines cardiac involvement, although it is usually subclinical. The alteration of cytokine network, especially the elevated levels of tumor necrosis factor-alpha, could be implicated in the cardiac alterations because it was observed, as for raised oxidative stress, in other heart diseases.

[Budd-Chiari syndrome with fatal outcome in a patient with polycythemia vera and antiphospholipid antibody syndrome]. / Sindrome di Budd-Chiari a decorso fatale in paziente con policitemia vera e sindrome da anticorpi antifosfolipidi.

We report the case of a 41-year-old woman, affected by Vaquez syndrome, admitted to our hospital for a severe pain in the right hypochondrium, suddenly followed by hepatomegaly and ascites. The clinical and laboratory data were suggestive of hepatic insufficiency and abdominal ultrasonography, integrated by color Doppler and computed tomography, revealed an interrupted hepatic venous outflow. In addition a spontaneous prolonged partial thromboplastin time was present and the patient was found to be positive for lupus anticoagulant. A transient clinical improvement, with a partial reperfusion of suprahepatic veins, was achieved with medical treatment by using anticoagulants, diuretics and paracentesis. However, the patient showed a subsequence of suprahepatic venous thrombosis, although two transjugular intrahepatic portosystemic shunts with stent placement and local thrombolysis were performed. The polycythemia vera is a disease mainly associated with Budd-Chiari syndrome but, in our patient, the thrombotic event occurred in spite of normal values of hematocrit and platelet count. Certainly in this case the lupus anticoagulant positivity represents an additional thrombogenic factor. Nowadays the antiphospholipid antibody syndrome is a recognized and not unusual cause of Budd-Chiari syndrome but, to our knowledge, this is the first case characterized by the presence of polycythemia vera and antiphospholipid antibody syndrome to be reported.