Characterization of human costal cartilage: is it an adapt tissue as graft for articular cartilage repair?

Several techniques and different biological or artificial tissues have been proposed as graft to restore articular defects. However, among the numerous and heterogeneous procedures proposed over time, the current literature findings are not conclusive. The aim of the current study is to evaluate if human costal cartilage can be suitable as graft for restoring articular cartilage defects. Knee articular cartilage and costal cartilage samples were obtained respectively from patients that underwent anterior cruciate ligament reconstruction (samples from notch plasty) or knee joint replacement and ear reconstruction or rhinoplasty through rib graft. The samples were stained with hematoxylin eosin, safranine-O, Gomori paraldehyde-fuchsin and Von Kossa for light microscopy. Immunohistochemistry was performed using anti-collagen I, II, IV and anti-SOX9 antibodies. Furthermore, samples were analyzed by transmission electron microcopy (TEM). In both cartilage, the cells are arranged in quite similar layers and the matrix show the same hyaline appearance: presence of type II collagen and solphated glycosaminoglycans, and absence of type I collagen and SOX-9. The bigger difference between the two hyaline tissues is the presence of perichondrium that surrounds all the specimens of costal cartilage. It consists of two separate layers where the inner one seems to get thinner with aging. The results show that rib cartilage seems to be an adapt tissue as graft for articular cartilage repair from a histological point of view. However, to date its therapeutic potential remains to be clearly defined by animal and clinical studies.

Potential of acetaminophen on the sublingual microcirculation and peripheral tissue perfusion of febrile septic patients: prospective observational study.

BACKGROUND: Acetaminophen (ACT) has been studied in septic patients with detectable plasmatic levels of cell-free hemoglobin (Hb), where it demonstrated to inhibit the hemoprotein-mediated lipid peroxidation and oxidative injury, with a potential of beneficial effect on the endothelium. On the basis of this background, the aim of this study was to evaluate the sublingual microcirculation and the peripheral tissue perfusion before-and-after administration of ACT on clinical judgment in a cohort of febrile septic and septic shock patients. METHODS: Prospective observational study. 50 adult septic and septic shocks treated with ACT for pyrexia, where the sublingual microcirculation and the peripheral tissue perfusion with Near Infrared Spectroscopy (NIRS) and vascular occlusion test (VOT) were evaluated before ACT (t0), after 30 min (t1) and after 2 h (t2). Cell-free Hb and the markers of oxidative stress and endothelial damage were measured at t0 and t2. RESULTS: The study showed a significant increase of the density of the perfused small and total vessels of the sublingual microcirculation 30 min after the infusion of ACT; it also showed an increase of the Microvascular Flow Index (MFI) and a decrease in the heterogeneity of the flow. At a peripheral muscular level, we found an acceleration in the reperfusion curve after VOT at t1, expression of a higher reactivity of the microvasculature. CONCLUSIONS: ACT infusion did not show a clear correlation with cell-free Hb; however, it exhibited protective effect toward the microcirculation that was evident in particular in septic patients. This correlation merits further exploration.

Dexamethasone affects Fas- and serum deprivation-induced cell death of human osteoblastic cells through survivin regulation.

Glucocorticoid-induced bone loss is the most prevalent form of secondary osteoporosis. Such loss could be due to the alteration of osteoclast and osteoblast lifespan through regulated apoptosis. The current study investigated the effect of dexamethasone on Fas- and starvation-induced apoptosis of mature osteoblasts and their precursors. Using the human osteoblastic hFOB1.19 and the MG63 osteosarcoma cell lines, we found that sub-lethal doses of dexamethasone act on pre-osteoblasts but not on mature cells by increasing their susceptibility to apoptosis. Apoptosis occurs in a caspase-dependent manner as both DNA fragmentation and mitochondrial transmembrane potential dissipation (ΔΨm) are inhibited by the pan-caspase inhibitor zVAD. The increased susceptibility of osteoblast precursors to apoptosis could be due to dexamethasonemediated down-regulation of survivin expression. Dexamethasone can up-regulate survivin, and to a lesser extent Bcl-2, in mature cells but not in pre-osteoblasts. In addition, it can induce FLIP over-expression in osteosarcoma cells. All these effects are inhibited by the glucocorticoid antagonist RU486, indicating that dexamethasone action is specific and, furthermore, that it depends on glucocorticoid receptor. Finally, we have found that survivin and Bcl-2 are essential for pre- and mature osteoblast survival as their silencing is sufficient to induce spontaneous apoptosis in both cell types. In conclusion, our data outline a new molecular mechanism of glucocorticoid-mediated bone loss due to the enhanced apoptosis of precursors compared to mature osteoblasts. Furthermore, the data suggest a mechanism of dexamethasone-induced resistance of osteosarcoma cells to Fas- and stress-induced apoptosis.

SARS-CoV-2 identification in lungs, heart and kidney specimens by transmission and scanning electron microscopy.

From two COVID-19-related deaths, samples of lung, heart and kidney were collected and processed for Transmission and Scanning Electron Microscopy (TEM and SEM) with the aim of identifying the virus. Virions of SARS-CoV-2 were found in all tissues by TEM and SEM, corroborating the hypothesis that the virus enters the cells of different organs. This is the first report identifying SARS-CoV-2 in different human tissues by TEM and SEM.

RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes.

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64-69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP-RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP-RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

Location of the binding site of the mannose-specific lectin comitin on F-actin.

We have used electron microscopy and computer image processing to produce a three-dimensional reconstruction of F-actin filaments decorated with the putative lectin and actin-binding protein comitin. These reconstructions show that comitin binds to F-actin at high radius primarily to actin subdomain 1. This location is distinctly different from the binding site on F-actin for other actin bundling proteins, such as members of the alpha-actinin family, and may result from the positively charged comitin interacting with negatively charged sites near the actin N terminus in subdomain 1. The location of the comitin binding site and its restriction to subdomain 1 on a single actin monomer is consistent with comitin's having a function distinct from other actin-binding proteins and, for example, would enable comitin to link bundled actin filaments to the Golgi.

Rat motor neuron plasticity induced by dorsal rhizotomy.

The control of peripheral structural plasticity of motor neurons by primary sensory neurons was studied in rat extensor digitorum longus (EDL) muscle. Polyinnervation of muscle fibers, sprouting and the motor neuron peripheral field size following L4 dorsal root cutting were evaluated using three different approaches: intracellular recording of end plate potentials, histochemical demonstration of sprouting and polyinnervation and in vivo recording of nerve-evoked twitch. Nodal sprouting was found in rhizotomized rats but not in controls and consistently muscle polyinnervation appeared. The muscle portion innervated by L3 ventral root was relatively reduced and that innervated by L5 was relatively enlarged: a trend to caudal shift of muscle innervation arose in rhizotomized rats. A control of motor neuron plasticity by primary sensory neurons is suggested.

Low FasL levels promote proliferation of human bone marrow-derived mesenchymal stem cells, higher levels inhibit their differentiation into adipocytes.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into several cell types. Bone marrow (BM)-MSCs mainly differentiate into osteoblasts or adipocytes. MSC interactions with their microenvironment directly affect their self-renewal/differentiation program. Here, we show for the first time that Fas ligand (FasL), a well-explored proapoptotic cytokine, can promote proliferation of BM-derived MSCs in vitro and inhibits their differentiation into adipocytes. BM-MSCs treated with a low FasL dose (0.5 ng/ml) proliferated more rapidly than untreated cells without undergoing spontaneous differentiation or apoptosis, whereas higher doses (25 ng/ml) induced significant though not massive BM-MSC death, with surviving cells maintaining a stem cell phenotype. At the molecular level, 0.5 ng/ml FasL induced ERK1/2 phosphorylation and survivin upregulation, whereas 25 ng/ml FasL induced caspase activation. Importantly, 25 ng/ml FasL reversibly prevented BM-MSC differentiation into adipocytes by modulating peroxisome proliferator-activated receptor gamma (PPARγ) and FABP4/aP2 expression induced by adipogenic medium. All such effects were inhibited by anti-Fas neutralizing antibody. The in vitro data regarding adipogenesis were confirmed using Fas(lpr) mutant mice, where higher PPARγ and FABP4/aP2 mRNA and protein levels were documented in whole tibia. These data show for the first time that the FasL/Fas system can have a role in BM-MSC biology via regulation of both proliferation and adipogenesis, and may have clinical relevance because circulating Fas/FasL levels decline with age and several age-related conditions, including osteoporosis, are characterized by adipocyte accumulation in BM.

[Presynaptic effects induced by pretreatment with formamide of the neuromuscular junction of the mouse]. / Effetti presinaptici indotti dal pretrattamento con formamide sulla guinzione neuromuscolare di topo.

Some techniques to block muscular nerve evoked contraction involve pharmacological approaches using synaptic blocking agents. Such methods interfere with normal synaptic transmission, and could introduce artifacts making difficult the experimental interpretation. The method based on the use of formamide pre-treatment should not interfere with synaptic physiology, indeed previous works suggest that the mechanism involved in block of muscle activity could depend on the decrease in specific postsynaptic membrane capacitance, and on the disruption of the morphology of the transverse tubule system. To prove this assumption we evaluated before and after formamide pre-treatment, some pre and postsynaptic parameters related to the spontaneous quantal release (MEPC). By means of the Loose patch clamp technique, we demonstrated, that formamide pre-treatment increases in an irreversible manner the frequency of spontaneous quantal release. Morphology of MEPC appear not modified by formamide pretreatment, which does not interfere with postsynaptic cholinergic receptors activity.

The effect of alpha 1 adrenoceptor antagonist prazosin on capillary supply, blood flow and performance in a rat model of chronic muscle ischaemia.

OBJECTIVE: This study explores whether the a1 blocker prazosin improves capillarisation, blood flow and muscle performance in chronically ischaemic muscles. DESIGN: Rat skeletal muscles were made ischaemic by unilateral ligation of the common iliac artery. The animals received prazosin and muscle blood flow, performance and capillary supply were studied 2 or 5 weeks later. MATERIALS AND METHODS: Prazosin (50 mg/l) was given in drinking water to animals with limited or intact blood supply. Muscle isometric twitch tension and fatigue index and blood flow at rest and during contractions (estimated by radio labelled microspheres) were measured in tibialis anterior, TA, and extensor digitorum longus, EDL. Capillary supply (capillary/fibre ratio, C/F) was estimated in frozen muscle sections stained for alkaline phosphatase in TA, EDL and soleus, S. RESULTS: Prazosin increased significantly capillary supply in ischaemic muscles after 2 and 5 weeks and blood flow during contractions (control muscles 163 +/- 6 ml. 100 g-1 min-1; ligated 2 weeks 25 +/- 7, ligated +prazosin 116 +/- 38 ; ligated 5 weeks 27 +/- 7, +prazosin 137 +/- 29, p < 0.05 vs. ligated). Fatigue index (final/peak tension over 5 min contractions at 4 Hz in %) was significantly improved by prazosin after 2 weeks (FI = 69 +/- 3% controls, 35 +/- 4% ligation, 60 +/- 3% ligation +prazosin) and less after 5 weeks (52 +/- 12% ligation, 61 +/- 3% ligation +prazosin). In contrast, prazosin had no effect on muscle performance in normal muscles in spite of it increased C/F and blood flow. CONCLUSIONS: Alpha1 adrenoceptor blockade is an effective way for improvement of capillary supply, blood flow and muscle performance in ischaemic muscles; the effect on muscle performance is most pronounced 2 weeks after induction of ischaemia.

Determination of high-energy phosphate compounds and inorganic phosphate by reversed-phase high-performance liquid chromatography: evaluation of myocardial metabolic status in aerobically perfused and hypoxic mouse heart.

The present paper describes a simple HPLC method designed for measuring high-energy phosphate (HEP) compounds in a single run and inorganic phosphate (Pi) in an other short run under the same HPLC conditions. Inorganic phosphate was estimated by using thymidine phosphorylase (EC 2.4.2.4) which catalyzes a reaction involving inorganic phosphate to produce 2-deoxyribose 1-phosphate and thymine. The thymine/Pi stoichiometry was 1. The method provides a reproducible instrument for evaluating myocardial high-energy metabolism under physiological and pathological conditions.

Reversible binding of glycolytic enzymes and size change in the actin-containing filaments of the frog skeletal muscle.

Electron microscopy was used in order to study the change of the actin-containing filaments when the bound glycolytic enzymes were removed by a high ionic strength solution. The effectiveness of the extraction medium was checked by estimating the aldolase activity released. Evidence was provided that the larger diameter of the actin filaments in the I-bands in comparison with the A-bands can be accounted for by the bound glycolytic enzymes.

In vivo pericyte-endothelial cell interaction during angiogenesis in adult cardiac and skeletal muscle.

Pericytes (PC) exert an inhibitory effect on endothelial cell (EC) proliferation in vitro and withdrawal of PC occurs prior to EC proliferation during pathological capillary growth in vivo. Using stereological analyses of PC and capillary EC fine structure, we have studied their relationship during the early stages of physiological angiogenesis using two in vivo models. In rat skeletal muscle where capillary growth was induced by indirect electrical stimulation, there was a reduction in the relative area of contact between pericytes and the capillary abluminal surface (22% vs 30% for normal controls; P < 0.05) and pericyte surface: volume ratio (12.5 vs 14.9 microns-1 for normal controls; P < 0.05) after 3 days of stimulation, at a time prior to the appearance of new capillaries. Further withdrawal of pericyte processes was evident at the point where an actual increase in capillary numbers was observed (7 days stimulation; PC surface: volume ratio = 10.4 microns-1), although the degree of PC-EC interdigitation increased. Similar changes, but to a lesser extent, were observed in both left ventricular myocardium and papillary muscles of pigs following 4-5 weeks chronic heart rate reduction. Although PC coverage of capillaries in the heart was found to be less than that in skeletal muscle, the relative contact area between PC and capillaries also showed a reduction in paced vs sham-operated control heart (papillary: 15% vs 20%; ventricle: 12% vs 16%; P < 0.05). Interdigitation of PC and EC was absent in cardiac muscle. These data suggest that retraction of PC may play a permissive role in controlling angiogenesis in vivo in normal adult tissue.

Gamma irradiation can reduce muscle damage in mdx dystrophic mice.

We report the effects of a single gamma irradiation delivered to the soleus muscle of one limb of normal and mdx mice at the age of 16-20 days. At 45, 75 and 90 days of age transverse cryostat sections from the mid-belly of the muscles were used for microscopic examination. In normal mice the growth of fibres was appreciably reduced by irradiation without fibre loss. In the irradiated soleus of mdx mice the number of the regenerated centrally nucleated fibres was very small and the total number of fibres was remarkably reduced. The number of the peripherally nucleated fibres, presumably surviving since the birth of the animal, was almost consistently larger than in the contralateral non-irradiated limb. The cross-sectional area of the irradiated fibres was smaller. It is well known that proliferation and fusion of satellite cells are required both for regeneration after fibre damage and for the normal postnatal growth of muscle fibres: irradiation appears to reduce regeneration and growth. It is suggested that irradiation reduces damage by reducing fusion associated with growth. Our hypothesis indirectly indicates a significant link between dystrophin deficiency and fibre necrosis and accounts well for many features of mouse dystrophy under natural and experimental conditions.