Experimental measurements and CFD modelling of hydroxyapatite scaffolds in perfusion bioreactors for bone regeneration

In the field of bone tissue engineering, particular interest is devoted to the development of 3D cultures to study bone cell proliferation under conditions similar to in vivo ones, e.g. by artificially producing mechanical stresses promoting a biological response (mechanotransduction). Of particular relevance in this context are the effects generated by the flow shear stress, which governs the nutrients delivery rate to the growing cells and which can be controlled in perfusion reactors. However, the introduction of 3D scaffolds complicates the direct measurement of the generated shear stress on the adhered cells inside the matrix, thus jeopardizing the potential of using multi-dimensional matrices. In this study, an anisotropic hydroxyapatite-based set of scaffolds is considered as a 3D biomimetic support for bone cells deposition and growth. Measurements of sample-specific flow resistance are carried out using a perfusion system, accompanied by a visual characterization of the material structure. From the obtained results, a subset of three samples is reproduced using 3D-Computational Fluid Dynamics (CFD) techniques and the models are validated by virtually replicating the flow resistance measurement. Once a good agreement is found, the analysis of flow-induced shear stress on the inner B-HA structure is carried out based on simulation results. Finally, a statistical analysis leads to a simplified expression to correlate the flow resistance with the entity and extensions of wall shear stress inside the scaffold. The study applies CFD to overcome the limitations of experiments, allowing for an advancement in multi-dimensional cell cultures by elucidating the flow conditions in 3D reactors

Neuronal Antibodies and Brain alterations in APECED Patients

APECED (Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Distrophy) is a rare autosomal recessive disorder. We previously found that sera samples from 9/14 patients revealed autoantibodies (Auto-Abs) reacting with cerebellum (GABAergic cells, n=5) and substantia nigra (SN; dopaminergic cells, n=5) [1]. Follow-up of the large majority of these patients was perfomed at least 10 years after the previous investigation. Indeed, on these patients, and on control age-matched subjects (n=14), we performed brain examinations using an MRI scanner. Obtained images were used to evaluate the volumes of white and gray matter (W.M and G.M., respectively) as well as the ventricles (III and IV). In addition, we used immunohistochemistry (IHC) on tissues from rat brain (after perfusion with 4% paraformaldehyde) in order to confirm the previous immunoreactivities or found new Auto-Abs cell targets. The brain MR revealed a reduction of G.M (p = 0.042) and cerebellum (p = 0.0012), and an increase of ventricles (p = 0.0001), compared to controls. Through IHC, after 10 years, we found 11/14 patients producing Auto-Abs against different brain neuronal cells. In detail, among the patients previously investigated and containing Auto-Abs against GABAergic perikarya in the cerebellum, 3 still contained the same immunoreactivity while 1 was unavailable, and 1 lost the reactivity. Instead, as to Auto-Abs against dopaminergic perikarya in the SN, 4 patients confirmed their previous reactivity, while 3 previously negative patients, revealed novel positivity (in total, n=7). A new immunoreactivity against the 5HT cells in the brainstem were also revealed in the same patients with Auto-Abs to SN (n=7). In conclusion, the co-presence of brain volume changes and neuronal Auto-Abs in APECED patients could suggest an autoimmune manifestation at the brain level that should be taken in consideration

Brain-reactive autoantibodies in neuropsychiatric systemic lupus erythematosus

IntroductionThe pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined. MethodsTo identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s). ResultsWe enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis (MS, n=22), and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain (cortex, hippocampus, and cerebellum) using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence (OR 2.4; p = 0.047), intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients' sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients' sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies (e.g., anti-NR2, anti-P-ribosomal protein, antiphospholipid) investigated by ELISA assay, only the anti-& beta;2-glycoprotein-I (a & beta;2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies. ConclusionIn conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include & beta;2GPI

The Matter of Future Heritage

In 2018, for the first time, the University of Bologna’s Board of PhD in Architecture and Design Culture assigned second-year PhD students the task of developing and managing an international conference and publishing its works. The organisers of the first edition of this initiative – Giacomo Corda, Pamela Lama, Viviana Lorenzo, Sara Maldina, Lia Marchi, Martina Massari and Giulia Custodi – have chosen to leverage the solid relationship between the Department of Architecture and the Municipality of Bologna to publish a call having to do with the European Year of Cultural Heritage 2018, in which the Municipality was involved. The theme chosen for the call, The Matter of Future Heritage, set itself the ambitious goal of questioning the future of a field of research – Cultural Heritage (CH) – that is constantly being  redefined. A work that was made particularly complex in Europe by the development of the H2020 programme, where the topic entered, surprisingly, not as a protagonist but rather as an articulation of other subjects that in the vision of the programme seemed evidently more urgent and, one might say, dominant. The resulting tensions have been considerable and with both negative and positive implications, all the more evident if we refer to the issues that are closest to us namely the city and the landscape

Brain-reactive autoantibodies in neuropsychiatric systemic lupus erythematosus

IntroductionThe pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) is widely unknown, and the role of autoantibodies is still undetermined.MethodsTo identify brain-reactive autoantibodies possibly related to NPSLE, immunofluorescence (IF) and transmission electron microscopy (TEM) on rat and human brains were performed. ELISA was used to reveal the presence of known circulating autoantibodies, while western blot (WB) was applied to characterize potential unknown autoantigen(s).ResultsWe enrolled 209 subjects, including patients affected by SLE (n=69), NPSLE (n=36), Multiple Sclerosis (MS, n=22), and 82 age- and gender-matched healthy donors (HD). Autoantibody reactivity by IF was observed in almost the entire rat brain (cortex, hippocampus, and cerebellum) using sera from NPSLE and SLE patients and was virtually negative in MS and HD. NPSLE showed higher prevalence (OR 2.4; p = 0.047), intensity, and titer of brain-reactive autoantibodies than SLE patients. Most of the patient sera with brain-reactive autoantibodies (75%) also stained human brains. Double staining experiments on rat brains mixing patients’ sera with antibodies directed against neuronal (NeuN) or glial markers showed autoantibody reactivity restricted to NeuN-containing neurons. Using TEM, the targets of brain-reactive autoantibodies were located in the nuclei and, to a lesser extent, in the cytoplasm and mitochondria. Given the high degree of colocalization between NeuN and brain-reactive autoantibodies, we assumed NeuN was a possible autoantigen. However, WB analysis with HEK293T cell lysates expressing or not expressing the gene encoding for NeuN protein (RIBFOX3) showed that patients’ sera carrying brain-reactive autoantibodies did not recognize the NeuN corresponding band size. Among the panel of NPSLE-associated autoantibodies (e.g., anti-NR2, anti-P-ribosomal protein, antiphospholipid) investigated by ELISA assay, only the anti-β2-glycoprotein-I (aβ2GPI) IgG was exclusively found in those sera containing brain-reactive autoantibodies.ConclusionIn conclusion, SLE and NPSLE patients possess brain-reactive autoantibodies but with higher frequency and titers found in NPSLE patients. Although many target antigens of brain-reactive autoantibodies are still undetermined, they likely include β2GPI

The hypothalamic-pituitary axis and autoantibody related disorders

This review summarized different studies reporting the presence of autoantibodies reacting against cells of the pituitary (APAs) and/or hypothalamus (AHAs). Both APAs and AHAs have been revealed through immunofluorescence using different kinds of substrates. Autoantibodies against gonadotropic cells were mainly found in patients affected by cryptorchidism and hypogonadotropic hypogonadism while those against prolactin cells were found in different kinds of patients, the majority without pituitary abnormalities. APAs to growth hormone (GH) cells have been associated with GH deficiency while those against the adrenocorticotropic cells have distinguished central Cushingâ\u80\u99s disease patients at risk of incomplete cure after surgical adenoma removal. AHAs to vasopressin cells have identified patients at risk of developing diabetes insipidus. APAs have been also found together with AHAs in patients affected by idiopathic hypopituitarism, but both were also present in different kinds of patients without abnormalities of the hypothalamic-pituitary axis. Despite some data being promising, the clinical use of pituitary and hypothalamus autoantibodies is still limited by the low diagnostic sensitivity, irreproducibility of the results, and the absence of autoantigen/s able to discriminate the autoimmune reaction involving the pituitary or the hypothalamus from the other autoimmune states

Protein-Based Capacitive Biosensors: a New Tool for Structure-Activity Relationship Studies

The present work reports a new application of a protein-based capacitive biosensor as an in vitro assay for the selectivity study of the bacterial periplasmic protein MerP and four MerP variants. The modified MerP proteins were produced by site-directed mutagenesis of the heavy metal associated motif (HMA). The MerP and modified MerPs selectivity for copper, zinc, cadmium and mercury bivalent ions were investigated and compared. The variations in the proteins affinity were related to the primary structure of the HMA motifs. Key amino acids for copper coordination of metalloproteins that contain the metal binding sequence Gly-Met-Thr-Cys-xxx-xxx-Cys were identified. The results brought insights valid for Menkes and Wilson ATPases. The protein-based capacitive biosensors were a simple and useful tool for studying structure-activity relationships of proteins

Reduction of Total Brain and Cerebellum Volumes Associated With Neuronal Autoantibodies in Patients With APECED

Context: In autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autoantibodies (AutoAbs) labeling brain neurons were reported; conversely, brain MRI alterations associated with these AutoAbs were never reported.Objectives: To describe brain alterations in APECED and to correlate them with AutoAbs against glutamic acid decarboxylase (GAD)-, tyrosine hydroxylase (TH)-, and 5-tryptophan hydroxylase (5-HT)- neurons. Design and participants: Fourteen Sardinian APECED patients and age-matched control subjects were recruited for MRI analysis and blood sampling to detect AutoAbs to GAD-, TH-, and 5-HT- neurons using rat brain sections. The majority of patients (n=12) were investigated for AutoAbs a decade ago and 7/12 were positive for AutoAbs to GAD and TH neurons. Main outcomes: APECED patients had smaller cerebellum and gray matter volumes, with a ventricular enlargement and a total liquor increase, compared to controls (p<0.01). In 11/14 patients, brain abnormalities were associated with AutoAbs to GAD and/or TH neurons (titer 1:100–15000), that had persisted for 10 years in 7/11 patients. AutoAbs to 5-HT neurons were revealed in all patients with AutoAbs to TH neurons. A decrease in whole brain and cerebellum volumes (p=0.028) was associated with AutoAbs to GAD neurons while a liquor increase with AutoAbs to GAD- and TH/5-HT neurons (p<0.05). HLA alleles did not appear to be involved in neuronal autoimmunity. Conclusions: For the first time, brain alterations together with neuronal AutoAbs were observed in 78.6% Sardinian APECED patients, suggesting a brain autoimmune reaction. Prolonged clinical follow-up must be conducted for the possible appearance of clinical neurological consequences. We revealed that APECED patients had smaller cerebellum and gray matter volumes, with a ventricular enlargement and a total liquor increase, all associated (in 11/14) with brain neuron AutoAbs

Relationship Between The First Diagnostic Ecg And Time To The Start Of Symptoms In Acute Myocardial Infarction

Q wave is typically a late finding on ECG during STEMI and consequently, when Q wave is found on the first ECG, many patients do not receive reperfusion therapy. In reality, the Q wave can also appear early in the course of the infarct and may represent a large reversible myocardial damage. The aim of our study was to evaluate the association between ischemic time and the appearance of the Q wave in STEMI acute phase and to assess whether the presence of an early Q wave can affect the management of STEMI patients. Materials and Methods: From January 1st 2009 to December 31st 2014, 248 consecutive patients with STEMI were transported from Emergency System to our CCU. Patients were divided into two groups based on the presence of Q wave in infarction leads at first ECG: group Q-wave (QWG) with 44 patients and group No Q-wave (NQWG) composed of 204 patients. For each patient following intervals were calculated: - the time between pain onset and first ECG (pain to ECG time ); - the time between the first ECG and the first intervention that restored the necrosis vessel patency (first medical contact-to-balloon time, FMC);- The time from the arrival of the patient to the hospital, and the reopening of the necrosis vessel (door-to-balloon time, DTB). Results: Pain to ECG time was greater in patients with Q-wave (119 ± 97 vs 113.02 ± 92.6 minutes, p = 0:09). The percentage of patients with early Q-wave increased progressively with increasing pain to ECG time from 2.5% in patients with Pain to ECG less than 30 minutes to 11.6% and 18.2% respectively in patients with Pain-to-ECG more than two hours and four hours (p for trend = 0.011). The DTB time, was quite comparable in both groups, 55 ± 54 vs 24.6 ± 31.2 min, p = 0.9. The FMC did not present statistically significant differences between groups (102.2 ± 35.9 minutes vs 97.8 ± 40.05 minutes, p = 0.48). Conclusions: The appearance of the Q wave is a time dependent phenomenon and is therefore affected by the delay between the onset of ischemia and the performing ECG, with a tendency to be present even in the earliest stages of the infarct itself. Despite the presence of Q waves at the first diagnostic ECG, management standard protocol of STEMI was applied in all patients, ensuring the similar reperfusion times