Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

The dystrophin gene, located at Xp21, codifies dystrophin, which is part of a protein complex responsible for the membrane stability of muscle cells. Its absence on muscle causes Duchenne Muscular Dystrophy (DMD), a severe disorder, while a defect of muscle dystrophin causes Becker Muscular Dystrophy (DMB), a milder disease. The replacement of the defective muscle through stem cells transplantation is a possible future treatment for these patients. Our objective was to analyze the potential of CD34+ stem cells from umbilical cord blood to differentiate in muscle cells and express dystrophin, in vitro. Protein expression was analyzed by Immunofluorescence, Western Blotting (WB) and Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). CD34+ stem cells and myoblasts from a DMD affected patient started to fuse with muscle cells immediately after co-cultures establishment. Differentiation in mature myotubes was observed after 15 days and dystrophin-positive regions were detected through Immunofluorescence analysis. However, WB or RT-PCR analysis did not detect the presence of normal dystrophin in co-cultures of CD34+ and DMD or DMB affected patients' muscle cells. In contrast, some CD34+ stem cells differentiated in dystrophin producers' muscle cells, what was observed by WB, reinforcing that this progenitor cell has the potential to originate muscle dystrophin in vitro, and not just in vivo like reported before.

Correlation analysis of the long latency auditory evoked potential N2 and cognitive P3 with the level of lead poisoning in children

Introduction: The effects of lead on children’s health have been widely studied. Aim: To analyze the correlation between the long latency auditory evoked potential N2 and cognitive P3 with the level of lead poisoning in Brazilian children. Methods: This retrospective study evaluated 20 children ranging in age from 7 to 14 years at the time of audiological and electrophysiological evaluations. We performed periodic surveys of the lead concentration in the blood and basic audiological evaluations. Furthermore, we studied the auditory evoked potential long latency N2 and cognitive P3 by analyzing the absolute latency of the N2 and P3 potentials and the P3 amplitude recorded at Cz. At the time of audiological and electrophysiological evaluations, the average concentration of lead in the blood was less than 10 ug/dL. Results: In conventional audiologic evaluations, all children had hearing thresholds below 20 dBHL for the frequencies tested and normal tympanometry findings; the auditory evoked potential long latency N2 and cognitive P3 were present in 95% of children. No significant correlations were found between the blood lead concentration and latency (p = 0.821) or amplitude (p = 0.411) of the P3 potential. However, the latency of the N2 potential increased with the concentration of lead in the blood, with a significant correlation (p = 0.030). Conclusion: Among Brazilian children with low lead exposure, a significant correlation was found between blood lead levels and the average latency of the auditory evoked potential long latency N2; however, a significant correlation was not observed for the amplitude and latency of the cognitive potential P3

Morphometric analysis of the placenta in the New World mouse Necromys lasiurus (Rodentia, Cricetidae): a comparison of placental development in cricetids and murids

Background: Stereology is an established method to extrapolate three-dimensional quantities from two-dimensional images. It was applied to placentation in the mouse, but not yet for other rodents. Herein, we provide the first study on quantitative placental development in a sigmodontine rodent species with relatively similar gestational time. Placental structure was also compared to the mouse, in order to evaluate similarities and differences in developmental patterns at the end of gestation. Methods: Fetal and placental tissues of Necromys lasiurus were collected and weighed at 3 different stages of gestation (early, mid and late gestation) for placental stereology. The total and relative volumes of placenta and of its main layers were investigated. Volume fractions of labyrinth components were quantified by the One Stop method in 31 placentae collected from different individuals, using the Mercator® software. Data generated at the end of gestation from N. lasiurus placentae were compared to those of Mus musculus domesticus obtained at the same stage. Results: A significant increase in the total absolute volumes of the placenta and its main layers occurred from early to mid-gestation, followed by a reduction near term, with the labyrinth layer becoming the most prominent area. Moreover, at the end of gestation, the total volume of the mouse placenta was significantly increased compared to that of N. lasiurus although the proportions of the labyrinth layer and junctional zones were similar. Analysis of the volume fractions of the components in the labyrinth indicated a significant increase in fetal vessels and sinusoidal giant cells, a decrease in labyrinthine trophoblast whereas the proportion of maternal blood space remained stable in the course of gestation. On the other hand, in the mouse, volume fractions of fetal vessels and sinusoidal giant cells decreased whereas the volume fraction of labyrinthine trophoblast increased compared to N. lasiurus placenta. Conclusions: Placental development differed between N. lasiurus and M. musculus domesticus. In particular, the low placental efficiency in N. lasiurus seemed to induce morphological optimization of fetomaternal exchanges. In conclusion, despite similar structural aspects of placentation in these species, the quantitative dynamics showed important differences.

Natural blood feeding and temperature shift modulate the global transcriptional profile of Rickettsia rickettsii infecting its tick vector

Rickettsia rickettsii is an obligate intracellular tick-borne bacterium that causes Rocky Mountain Spotted Fever (RMSF), the most lethal spotted fever rickettsiosis. When an infected starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. Using customized oligonucleotide microarrays and high-throughput microfluidic qRT-PCR, we analyzed the effects of a 10 degrees C temperature elevation and of a blood meal on the transcriptional profile of R. rickettsii infecting the tick Amblyomma aureolatum. This is the first study of the transcriptome of a bacterium in the genus Rickettsia infecting a natural tick vector. Although both stimuli significantly increased bacterial load, blood feeding had a greater effect, modulating five-fold more genes than the temperature upshift. Certain components of the Type IV Secretion System (T4SS) were up-regulated by blood feeding. This suggests that this important bacterial transport system may be utilized to secrete effectors during the tick vector's blood meal. Blood feeding also up-regulated the expression of antioxidant enzymes, which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. The modulated genes identified in this study, including those encoding hypothetical proteins, require further functional analysis and may have potential as future targets for vaccine development.

Dynamics of cell and tissue genesis in the male reproductive system of ticks (Acari: Ixodidae) Amblyomma cajennense (Fabricius, 1787) and Amblyomma aureolatum (Pallas, 1772): a comparative analysis

Ticks are classified into three families: Argasidae, Ixodidae, and Nutalliellidae. The taxonomy and phylogeny within Ixodidae are still discussed by the specialists, thus requiring further studies. Amblyomma cajennese and Amblyomma aureolatum (Brazil) belong to two species complexes known as “cajennese” and “ovale”, respectively, and are directly related to the transmission of the Brazilian spotted fever. This confirms the medical and veterinary significance of these species, as well as the need for further morphological studies that will bring a better understanding of their taxonomy, phylogeny, and control. In this context, the present study aimed to characterize the morphology of the male reproductive system of A. cajennese and A. aureolatum when unfed and after 4 days of feeding, thereby seeking to: (a) distinguish the two species or “complexes”, and (b) study an internal system which has the potential to be targeted by acaricides. Therefore, males from both species (unfed and after 4 days of feeding) were cold-anesthetized, dissected, and had their reproductive systems removed for histological analysis. The results showed that the morphology of the male reproductive system is generally similar between both species, like in other Ixodidae ticks, exhibiting a multilobed accessory gland complex related to seminal fluid secretion, a pair of vasa deferentia and a pair of testes housing germ cells (spermatocytes) in different stages. The main differences were found in the development of the accessory gland complex cells and germ cells, showing that the maturation of the male reproductive system starts later in A. aureolatum, when compared to A. cajennese. However, during the blood meal, A. aureolatum development is increased, thus making germ cell maturation and gland complex activity higher than in A. cajennese. This study shows the differences in the development of the male reproductive systems of both species, while providing information that can assist in the establishment of new control methods.

Quantitative analysis of photodynamic therapy effects in rat mammary tumor vascular density using image-pro plus software

Photodynamic therapy (PDT) is a treatment modality that has advanced rapidly in recent years. It causes tissue and vascular damage with the interaction of a photosensitizing agent (PS), light of a proper wavelength, and molecular oxygen. Evaluation of vessel damage usually relies on histopathology evaluation. Results are often qualitative or at best semi-quantitative based on a subjective system. The aim of this study was to evaluate, using CD31 immunohistochem- istry and image analysis software, the vascular damage after PDT in a well-established rodent model of chemically induced mammary tumor. Fourteen Sprague-Dawley rats received a single dose of 7,12-dimethylbenz(a)anthraxcene (80 mg/kg by gavage), treatment efficacy was evaluated by comparing the vascular density of tumors after treatment with Photogem® as a PS, intraperitoneally, followed by interstitial fiber optic lighting, from a diode laser, at 200 mW/cm and light dose of 100 J/cm directed against his tumor (7 animals), with a control group (6 animals, no PDT). The animals were euthanized 30 hours after the lighting and mammary tumors were removed and samples from each lesion were formalin-fixed. Immunostained blood vessels were quantified by Image Pro-Plus version 7.0. The control group had an average of 3368.6 ± 4027.1 pixels per picture and the treated group had an average of 779 ± 1242.6 pixels per area (P < 0.01), indicating that PDT caused a significant decrease in vascular density of mammary tumors. The combined immu- nohistochemistry using CD31, with selection of representative areas by a trained pathology, followed by quantification of staining using Image Pro-Plus version 7.0 system was a practical and robust methodology for vessel damage evalua- tion, which probably could be used to assess other antiangiogenic treatments.

Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery

Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures

CT perfusion image processing: analysis of liver tumors

Perfusion CT imaging of the liver has potential to improve evaluation of tumour angiogenesis. Quantitative parameters can be obtained applying mathematical models to Time Attenuation Curve (TAC). However, there are still some difficulties for an accurate quantification of perfusion parameters due, for example, to algorithms employed, to mathematical model, to patient’s weight and cardiac output and to the acquisition system. In this thesis, new parameters and alternative methodologies about liver perfusion CT are presented in order to investigate the cause of variability of this technique. Firstly analysis were made to assess the variability related to the mathematical model used to compute arterial Blood Flow (BFa) values. Results were obtained implementing algorithms based on “ maximum slope method” and “Dual input one compartment model” . Statistical analysis on simulated data demonstrated that the two methods are not interchangeable. Anyway slope method is always applicable in clinical context. Then variability related to TAC processing in the application of slope method is analyzed. Results compared with manual selection allow to identify the best automatic algorithm to compute BFa. The consistency of a Standardized Perfusion Index (SPV) was evaluated and a simplified calibration procedure was proposed. At the end the quantitative value of perfusion map was analyzed. ROI approach and map approach provide related values of BFa and this means that pixel by pixel algorithm give reliable quantitative results. Also in pixel by pixel approach slope method give better results. In conclusion the development of new automatic algorithms for a consistent computation of BFa and the analysis and definition of simplified technique to compute SPV parameter, represent an improvement in the field of liver perfusion CT analysis.

A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Flow Field–Flow Fractionation for size analysis and characterization of nanoparticles for applications in Life Sciences

Nanotechnologies are rapidly expanding because of the opportunities that the new materials offer in many areas such as the manufacturing industry, food production, processing and preservation, and in the pharmaceutical and cosmetic industry. Size distribution of the nanoparticles determines their properties and is a fundamental parameter that needs to be monitored from the small-scale synthesis up to the bulk production and quality control of nanotech products on the market. A consequence of the increasing number of applications of nanomaterial is that the EU regulatory authorities are introducing the obligation for companies that make use of nanomaterials to acquire analytical platforms for the assessment of the size parameters of the nanomaterials. In this work, Asymmetrical Flow Field-Flow Fractionation (AF4) and Hollow Fiber F4 (HF5), hyphenated with Multiangle Light Scattering (MALS) are presented as tools for a deep functional characterization of nanoparticles. In particular, it is demonstrated the applicability of AF4-MALS for the characterization of liposomes in a wide series of mediums. Afterwards the technique is used to explore the functional features of a liposomal drug vector in terms of its biological and physical interaction with blood serum components: a comprehensive approach to understand the behavior of lipid vesicles in terms of drug release and fusion/interaction with other biological species is described, together with weaknesses and strength of the method. Afterwards the size characterization, size stability, and conjugation of azidothymidine drug molecules with a new generation of metastable drug vectors, the Metal Organic Frameworks, is discussed. Lastly, it is shown the applicability of HF5-ICP-MS for the rapid screening of samples of relevant nanorisk: rather than a deep and comprehensive characterization it this time shown a quick and smart methodology that within few steps provides qualitative information on the content of metallic nanoparticles in tattoo ink samples.

Comprehensive analysis of lymph node stroma-expressed Ig superfamily members reveals redundant and nonredundant roles for ICAM-1, ICAM-2, and VCAM-1 in lymphocyte homing

Although it is well established that stromal intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule-1 (VCAM-1) mediate lymphocyte recruitment into peripheral lymph nodes (PLNs), their precise contributions to the individual steps of the lymphocyte homing cascade are not known. Here, we provide in vivo evidence for a selective function for ICAM-1 > ICAM-2 > VCAM-1 in lymphocyte arrest within noninflamed PLN microvessels. Blocking all 3 CAMs completely inhibited lymphocyte adhesion within PLN high endothelial venules (HEVs). Post-arrest extravasation of T cells was a 3-step process, with optional ICAM-1-dependent intraluminal crawling followed by rapid ICAM-1- or ICAM-2-independent diapedesis and perivascular trapping. Parenchymal motility of lymphocytes was modestly reduced in the absence of ICAM-1, while ICAM-2 and alpha4-integrin ligands were not required for B-cell motility within follicles. Our findings highlight nonredundant functions for stromal Ig family CAMs in shear-resistant lymphocyte adhesion in steady-state HEVs, a unique role for ICAM-1 in intraluminal lymphocyte crawling but redundant roles for ICAM-1 and ICAM-2 in lymphocyte diapedesis and interstitial motility.

Comparison of non-calibrated pulse-contour analysis with continuous thermodilution for cardiac output assessment in patients with induced hypothermia after cardiac arrest

Induced mild hypothermia after cardiac arrest interferes with clinical assessment of the cardiovascular status of patients. In this situation, non-invasive cardiac output measurement could be useful. Unfortunately, arterial pulse contour is altered by temperature, and the performance of devices using arterial blood pressure contour analysis to derive cardiac output may be insufficient.

Sperm analysis of patients after successful treatment of childhood acute lymphoblastic leukemia with chemotherapy

Survivors of childhood acute lymphoblastic leukemia (ALL) treated with radiotherapy are at risk for impaired fertility. Whether chemotherapy alone is also long-term gonadotoxic is unclear. We assessed gonadal function in 11 male ALL-survivors treated with the same chemotherapy regimen and compared sperm analysis to healthy men. While sex hormone levels were normal in all subjects, 5/11 survivors showed pathological sperm concentration and 4/11 a decreased total sperm count compared to WHO criteria. Compared to healthy controls, all quantitative parameters in semen analysis of survivors were decreased. This suggests that treatment with chemotherapeutic agents alone, even in moderate doses, might have a gonadotoxic effect.

Hair analysis for Delta9-tetrahydrocannabinolic acid A--new insights into the mechanism of drug incorporation of cannabinoids into hair

Differentiation between external contamination and incorporation of drugs or their metabolites from inside the body via blood, sweat or sebum is a general issue in hair analysis and of high concern when interpreting analytical results. In hair analysis for cannabinoids the most common target is Delta9-tetrahydrocannabinol (THC), sometimes cannabidiol (CBD) and cannabinol (CBN) are determined additionally. After repeated external contamination by cannabis smoke these analytes are known to be found in hair even after performing multiple washing steps. A widely accepted strategy to unequivocally prove active cannabis consumption is the analysis of hair extracts for the oxidative metabolite 11-nor-9-carboxy-THC (THC-COOH). Although the acidic nature of this metabolite suggests a lower rate of incorporation into the hair matrix compared to THC, it is not fully understood up to now why hair concentrations of THC-COOH are generally found to be much lower (mostly <10 pg/mg) than the corresponding THC concentrations. Delta9-Tetrahydrocannabinolic acid A (THCA A) is the preliminary end product of the THC biosynthesis in the cannabis plant. Unlike THC it is non-psychoactive and can be regarded as a 'precursor' of THC being largely decarboxylated when heated or smoked. The presented work shows for the first time that THCA A is not only detectable in blood and urine of cannabis consumers but also in THC positive hair samples. A pilot experiment performed within this study showed that after oral intake of THCA A on a regular basis no relevant incorporation into hair occurred. It can be concluded that THCA A in hair almost exclusively derives from external contamination e.g. by side stream smoke. Elevated temperatures during the analytical procedure, particularly under alkaline conditions, can lead to decarboxylation of THCA A and accordingly increase THC concentrations in hair. Additionally, it has to be kept in mind that in hair samples tested positive for THCA A at least a part of the 'non-artefact' THC probably derives from external contamination as well, because in condensate of cannabis smoke both THC and THCA A are present in relevant amounts. External contamination by side stream smoke could therefore explain the great differences in THC and THC-COOH hair concentrations commonly found in cannabis users.

Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS

Phosphatidylethanol (PEth) is an abnormal phospholipid carrying two fatty acid chains. It is only formed in the presence of ethanol via the action of phospholipase D (PLD). Its use as a biomarker for alcohol consumption is currently under investigation. Previous methods for the analysis of PEth included high-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD), which is unspecific for the different homologues--improved methods are now based on time of flight mass spectrometry (TOF-MS) and tandem mass spectrometry (MS/MS). The intention of this work was to identify as many homologues of PEth as possible. A screening procedure using multiple-reaction monitoring (MRM) for the identified homologues has subsequently been established. For our investigations, autopsy blood samples collected from heavy drinkers were used. Phosphatidylpropanol 16:0/18:1 (internal standard) was added to the blood samples prior to liquid-liquid extraction using borate buffer (pH 9), 2-propanol and n-hexane. After evaporation, the samples were redissolved in the mobile phase and injected into the LC-MS/MS system. Compounds were separated on a Luna Phenyl Hexyl column (50 mm x 2 mm, 3 microm) by gradient elution, using 2 mM ammonium acetate and methanol/acetone (95/5; v/v). A total of 48 homologues of PEth could be identified by using precursor ion and enhanced product ion scans (EPI).