Angiotensin I and II Stimulate Cell Invasion of SARS-CoV-2: Potential Mechanism via Inhibition of ACE2 Arm of RAS.

Angiotensin-converting enzyme 2 (ACE2), one of the key enzymes of the renin-angiotensin system (RAS), plays an important role in SARS-CoV-2 infection by functioning as a virus receptor. Angiotensin peptides Ang I and Ang II, the substrates of ACE2, can modulate the binding of SARS-CoV-2 Spike protein to the ACE2 receptor. In the present work, we found that co incubation of HEK-ACE2 and Vero E6 cells with the SARS-CoV-2 Spike pseudovirus (PVP) resulted in stimulation of the virus entry at low and high micromolar concentrations of Ang I and Ang II, respectively. The potency of Ang I and Ang II stimulation of virus entry corresponds to their binding affinity to ACE2 catalytic pocket with 10 times higher efficiency of Ang II. The Ang II induced mild increase of PVP infectivity at 20 microM; while at 100 microM the increase (129.74+/-3.99 %) was highly significant (p<0.001). Since the angiotensin peptides act in HEK ACE2 cells without the involvement of angiotensin type I receptors, we hypothesize that there is a steric interaction between the catalytic pocket of the ACE2 enzyme and the SARS-CoV-2 S1 binding domain. Oversaturation of the ACE2 with their angiotensin substrate might result in increased binding and entry of the SARS-CoV-2. In addition, the analysis of angiotensin peptides metabolism showed decreased ACE2 and increased ACE activity upon SARS-CoV-2 action. These effects should be taken into consideration in COVID-19 patients suffering from comorbidities such as the over-activated renin-angiotensin system as a mechanism potentially influencing the SARS-CoV-2 invasion into recipient cells.

Brain Fluid Channels for Metabolite Removal.

The adult human brain represents only 2% of the body's total weight, however it is one of the most metabolically active organs in the mammalian body. Its high metabolic activity necessitates an efficacious waste clearance system. Besides the blood, there are two fluids closely linked to the brain and spinal cord drainage system: interstitial fluid (ISF) and cerebrospinal fluid (CSF). The aim of this review is to summarize the latest research clarifying the channels of metabolite removal by fluids from brain tissue, subarachnoid space (SAS) and brain dura (BD). Special attention is focused on lymphatic vascular structures in the brain dura, their localizations within the meninges, morphological properties and topographic anatomy. The review ends with an account of the consequences of brain lymphatic drainage failure. Knowledge of the physiological state of the clearance system is crucial in order to understand the changes related to impaired brain drainage.

Bone Marrow-Derived Cells Participate in Composition of the Satellite Cell Niche in Intact and Regenerating Mouse Skeletal Muscle.

The cellular components of the satellite cell niche participate in the regulation of skeletal muscle regeneration. Beside myogenic cells at different developmental stages, this niche is formed by cells of the immune system, the interstitial connective tissue and the vascular system. Unambiguous determination of the origin of these cell types could contribute to optimization of the cell-based therapy of skeletal muscle disorders. In our work, we intravenously transplanted mouse GFP+ unseparated bone marrow cells into whole-body lethally irradiated immunocompetent mice four weeks before cardiotoxin-induced injury of the recipients' skeletal muscles. Seven and 28 days after the toxin injection, the injured regenerating and contralateral intact muscles were examined for identification of GFP+ bone marrow-derived cells by direct fluorescence, protein immunohistochemistry and immunogold transmission electron microscopy. In both the intact and injured muscles, GFP positivity was determined in immune cells, mainly in macrophages, and in interstitial spindleshaped cells. Moreover, in the injured muscles, rare GFP+ endothelial cells of the blood vessels and newly formed myotubes and muscle fibres were present. Our results confirmed the ability of bone marrowderived cells to contribute to the cellular component of the satellite cell niche in the intact and regenerating skeletal muscle. These cells originated not only from haematopoietic stem cells, but obviously also from other stem or progenitor cells residing in the bone marrow, such as multipotent mesenchymal stromal cells and endothelial progenitors.

Longitudinal enlargement of the lesion after spinal cord injury in the rat: a consequence of malignant edema?

STUDY DESIGN: Experimental animal study. OBJECTIVES: Quantitative analysis of secondary changes in lesion size after experimental spinal cord injury (SCI) in the rat, with special emphasis to the formation of dorsal column lesions. SETTING: Slovakia. METHODS: After SCI in the rat, animals survived for different periods ranging from 5 min to 7 days. Their whole spinal cords were cut transversally into 1 mm thick slabs. On each slab, the lesion profile was outlined. The overall shape of the lesion was reconstructed from a series of consecutive profiles and its length was measured. RESULTS: Immediately after injury, a spindle-shaped hemorrhagic contusive lesion was observed, with the length of ~15 mm. After a quiescent phase lasting for at least 1 h, there was a dramatic secondary enlargement of the lesion and its length increased up to 40 mm between 1 and 48 h. The fully developed lesion consisted of the spindle-shaped epicenter and long cranial and caudal protrusions located in the midline between dorsal columns. CONCLUSION: We propose that secondary enlargement of the lesion can be explained by posttraumatic swelling. The expanding tissues are pushed out in longitudinal axis along the mechanically weakest parts of the spinal cord. Additional data that support this hypothesis are presented. Our findings indicate that malignant posttraumatic edema might have an important role in pathomechanisms of secondary injury after SCI.

Delivery of Alginate Scaffold Releasing Two Trophic Factors for Spinal Cord Injury Repair.

Spinal cord injury (SCI) has been implicated in neural cell loss and consequently functional motor and sensory impairment. In this study, we propose an alginate-based neurobridge enriched with/without trophic growth factors (GFs) that can be utilized as a therapeutic approach for spinal cord repair. The bioavailability of key GFs, such as Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) released from injected alginate biomaterial to the central lesion site significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons (choline acetyltransferase positive motoneurons) and sensory fibres. In addition, we document enhanced outgrowth of corticospinal tract axons and presence of blood vessels at the central lesion. Tissue proteomics was performed at 3, 7 and 10 days after SCI in rats indicated the presence of anti-inflammatory factors in segments above the central lesion site, whereas in segments below, neurite outgrowth factors, inflammatory cytokines and chondroitin sulfate proteoglycan of the lectican protein family were overexpressed. Collectively, based on our data, we confirm that functional recovery was significantly improved in SCI groups receiving alginate scaffold with affinity-bound growth factors (ALG+GFs), compared to SCI animals without biomaterial treatment.

IT delivery of ChABC modulates NG2 and promotes GAP-43 axonal regrowth after spinal cord injury.

Chondroitin sulphate proteoglycans (CSPGs) with the major component NG2 have an inhibitory effect on regeneration of damaged axons after spinal cord injury. In this study, we investigate whether the digestion of CSPGs by chondroitinase ABC (ChABC) may decrease the NG2 expression and promote axon regrowth through the lesion site. Rats underwent spinal cord compression injury and were treated with ChABC or vehicle through an intrathecal catheter delivery at 2, 3, and 4 days after injury. In addition, animals were behaviorally scored using BBB test in weekly intervals after SCI. Based on immunocytochemical analyses, we have quantified distribution of NG2 glycoprotein and GAP-43 in spinal cord tissue in both experimental groups. Multiple injections of ChABC caused decrease of NG2 expression at lesion site at 5 and 7 days, but not at 14 and 28 days in comparison with vehicle-treated rats and significantly enhanced GAP-43 expression during the entire survival. The densitometry analysis showed significantly higher GAP-43 immunoreactivity (1.8-2.2-fold) in the regrowing axons and cell bodies within the central lesion cavity when compared with vehicle group. Longitudinally oriented and disorganized GAP-43-labeled axons were able to infiltrate and penetrate damaged tissue. The outgrowth of GAP-43 axons after CHABC delivery was significantly longer (≤0.457 mm) when compared with the length of axons in vehicle-treated rats (≤0.046 mm). Present findings suggest that degradation of NG2 with acute IT ChABC treatment may promote ongoing (long-lasting) axonal regenerative processes at late survival (14 and 28 days), but with no significant impact on the improvement of motor function.

Chondroitinase ABC treatment and the phenotype of neural progenitor cells isolated from injured rat spinal cord.

The aim of the present study was to investigate whether enzyme chondroitinase ABC (ChABC) treatment influences the phenotype of neural progenitor cells (NPCs) derived from injured rat spinal cord. Adult as well as fetal spinal cords contain a pool of endogenous neural progenitors cells, which play a key role in the neuroregenerative processes following spinal cord injury (SCI) and hold particular promise for therapeutic approaches in CNS injury or neurodegenerative disorders. In our study we used in vitro model to demonstrate the differentiation potential of NPCs isolated from adult rat spinal cord after SCI, treated with ChABC. The intrathecal delivery of ChABC (10 U/ml) was performed at day 1 and 2 after SCI. The present findings indicate that the impact of SCI resulted in a decrease of all NPCs phenotypes and the ChABC treatment, on the contrary, caused an opposite effect.

Role of transplanted bone marrow cells in response to skeletal muscle injury.

The recently discovered capacity of bone marrow cells (BMCs) to contribute to injury-induced skeletal muscle regeneration has brought new possibilities in the treatment of skeletal muscle diseases. However, a suitable method of BMC transplantation usable for such therapy has to be established. In this work, recipient mice were intramuscularly injected with cardiotoxin, then whole-body lethally irradiated to eradicate satellite cells in their injured tibialis anterior (TA) muscles and to suppress haematopoiesis, and subsequently intravenously transplanted with lacZ+ BMCs with the aim to investigate the role of exogenous BMCs in response to skeletal muscle injury. Seven to 33 days after grafting, recipient TA muscles were examined to detect donor-derived X-gal+ cells and analysed by quantitative PCR. In injured recipients' muscles, X-gal positivity was identified 14 and 33 days after grafting in some infiltrating neutrophils and macrophages, infrequently in fibroblasts of endomysium, and in many large multinucleated cells (devoid of myogenic markers desmin and nestin) resembling foreign body giant cells situated in the vicinity of necrotic muscle fibres. qPCR confirmed the presence of transplanted lacZ+ BMCs in injured recipients' muscles. Our results proved the ability of intravenously transplanted adult BMCs to settle in injured muscles and generate blood cells that infiltrated endomysium and took part in the cleaning reaction. After inhibition of endogenous myogenesis, BMCs were not able to participate in formation of new muscle fibres due to persisting necrosis of degenerated muscle fibres. Instead, BMCs attempted to resorb necrotic structures, which confirmed the indispensable role of bone marrow-derived macrophages in skeletal muscle regeneration.

Genetic structure and contrasting selection pattern at two major histocompatibility complex genes in wild house mouse populations.

The mammalian major histocompatibility complex (MHC) is a tightly linked cluster of immune genes, and is often thought of as inherited as a unit. This has led to the hope that studying a single MHC gene will reveal patterns of evolution representative of the MHC as a whole. In this study we analyse a 1000-km transect of MHC variation traversing the European house mouse hybrid zone to compare signals of selection and patterns of diversification at two closely linked MHC class II genes, H-2Aa and H-2Eb. We show that although they are 0.01 cM apart (that is, recombination is expected only once in 10 000 meioses), disparate evolutionary patterns were detected. H-2Aa shows higher allelic polymorphism, faster allelic turnover due to higher mutation rates, stronger positive selection at antigen-binding sites and higher population structuring than H-2Eb. H-2Eb alleles are maintained in the gene pool for longer, including over separation of the subspecies, some H-2Eb alleles are positively and others negatively selected and some of the alleles are not expressed. We conclude that studies on MHC genes in wild-living vertebrates can give substantially different results depending on the MHC gene examined and that the level of polymorphism in a related species is a poor criterion for gene choice.

Enrichment of rat oligodendrocyte progenitor cells by magnetic cell sorting.

The embryonic, neonatal, as well as adult rat spinal cords harbor a pool of neural stem cells (NSCs), which may be easily isolated and used to replace neuronal cell loss or remyelinate damaged axons following various neurodegenerative disorders. In the present study we have used magnetic cell sorting (MACs) technology to generate enriched oligodendroglial cell populations from the embryonic (E16) rat spinal cord. Target cells were separated by positive selection, using specific A2B5 antibody-labeled MicroBeads achieving optimal recovery and high purity of pro-oligodendroglial cells. Based on immunocytochemical analyses for oligodendroglial developmental markers (A2B5, NG2, RIP and MBP) we were able to characterize and quantify oligodendroglial progenitors (OPCs) and mature oligodendroglial cells in: (i) unseparated heterogeneous population of NSCs, or in (ii) antigen-antibody separated NSCs. Our results showed that MACs technology enable us to gain enriched OPCs from heterogeneous population of spinal NSCs, resulting in a 58-61% of mature oligodendrocytes content (MBP+, RIP+) in comparison to 6-12% of oligodendroglial cells acquired from unseparated population. In addition, the enriched OPCs could be cultured in vitro for several >8 passages, giving rise to a high number of newly formed spheres, as well as high expansion potential. These experiments indicate that MACs technology provide a feasible approach for experimental cell enrichment of desired oligodendroglial progeny, which may be used in future trials for cell-based therapies to treat spinal cord injury.

Re-expression of nestin in the myocardium of postinfarcted patients.

Intact cardiac muscle cells in the adult heart do not express intermediate filament nestin. In this study, we report on widespread expression of intermediate filament nestin in human myocardium of patients who died from the myocardial infarction. Nestin was detected in cardiomyocytes, endothelial cells, and few interstitial cells. Elevated levels of nestin were observed in cardiac muscle cells in all specimens, although the intensity of immunoreactivity and distribution of the signal differed. The strongest immunoreactivity was observed from 4 days after myocardial infarction in the infarction border zone where nestin was distributed homogeneously in the entire sarcoplasm of cardiac muscle cells. Within the following week, nestin in immunoreactive cardiomyocytes was redistributed and restricted to small subsarcolemmal foci and to intercalated discs. Angiogenic capillaries that grew between vital nestin-positive cardiomyocytes and entered the necrotic area expressed also high levels of nestin. Nestin-positive endothelial cells were often observed in mutual interactions with nestin-positive cardiac muscle cells. These findings document a crucial role of nestin in remodeling cytoskeleton of cells in the human postinfarcted myocardium.

Phenotypic and morphological characterization of in vitro oligodendrogliogenesis.

The neurosphere assay has been used to maintain neural progenitor cells (NPCs) in the undifferentiated state. These cells are multipotent and gave rise to neurons and glial cells. Here we show that within 10 days of culture, neurospheres contained precursors and differentiated progeny of all three major central nervous system (CNS) cell lineages and these occupied distinct zones. The microenvironment of the inner zone supported cell differentiation. Cells of oligodendroglial lineage generated within the neurosphere were frequently observed. Of these cells, A2B5(+) cells were homogeneously distributed in the neurospheres, NG2(+) cells preferentially occupied the outer zone and O4(+) cells were localized at the inner zone of 10 day-old neurospheres. We prevented a massive cell death of dissociated neurosphere cells seen after differentiation triggered with adhesion and fetal calf serum by adding epidermal growth factor and basic fibroblast growth factor to the culture medium. Under these conditions, less than one third of cells did not express cell specific markers, glial fibrillary acidic protein-positive astroglia represented 43.4%, NG2(+) and/or O4(+) oligodendroglia represented 24.3%, and betaIII-tubulin(+) neurons 3.1% of cells recovered after neurosphere differentiation. We present evidence that oligodendroglial cells differentiate in a stepwise process as a result of their distribution in subsets that represent distinct developmental stages according to antigenic and morphological criteria. These include oligodendrocyte progenitors, preoligodendrocytes, and oligodendrocytes. The highly complex morphology of mature oligodendrocytes was compatible with functional cells.

Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells.

Transient spinal cord ischemia in humans can lead to the development of permanent paraplegia with prominent spasticity and rigidity. Histopathological analyses of spinal cords in animals with ischemic spastic paraplegia show a selective loss of small inhibitory interneurons in previously ischemic segments but with a continuing presence of ventral alpha-motoneurons and descending cortico-spinal and rubro-spinal projections. The aim of the present study was to examine the effect of human spinal stem cells (hSSCs) implanted spinally in rats with fully developed ischemic paraplegia on the recovery of motor function and corresponding changes in motor evoked potentials. In addition the optimal time frame for cell grafting after ischemia and the optimal dosing of grafted cells were also studied. Spinal cord ischemia was induced for 10 min using aortic occlusion and systemic hypotension. In the functional recovery study, hSSCs (10,000-30,000 cells/0.5 mul/injection) were grafted into spinal central gray matter of L2-L5 segments at 21 days after ischemia. Animals were immunosuppressed with Prograf (1 mg/kg or 3 mg/kg) for the duration of the study. After cell grafting the recovery of motor function was assessed periodically using the Basso, Beattie and Bresnahan (BBB) scoring system and correlated with the recovery of motor evoked potentials. At predetermined times after grafting (2-12 weeks), animals were perfusion-fixed and the survival, and maturation of implanted cells were analyzed using antibodies recognizing human-specific antigens: nuclear protein (hNUMA), neural cell adhesion molecule (hMOC), neuron-specific enolase (hNSE) and synapthophysin (hSYN) as well as the non-human specific antibodies TUJ1, GFAP, GABA, GAD65 and GLYT2. After cell grafting a time-dependent improvement in motor function and suppression of spasticity and rigidity was seen and this improvement correlated with the recovery of motor evoked potentials. Immunohistochemical analysis of grafted lumbar segments at 8 and 12 weeks after grafting revealed intense hNSE immunoreactivity, an extensive axo-dendritic outgrowth as well as rostrocaudal and dorsoventral migration of implanted hNUMA-positive cells. An intense hSYN immunoreactivity was identified within the grafts and in the vicinity of persisting alpha-motoneurons. On average, 64% of hSYN terminals were GAD65 immunoreactive which corresponded to GABA immunoreactivity identified in 40-45% of hNUMA-positive grafted cells. The most robust survival of grafted cells was seen when cells were grafted 21 days after ischemia. As defined by cell survival and laminar distribution, the optimal dose of injected cells was 10,000-30,000 cells per injection. These data indicate that spinal grafting of hSSCs can represent an effective therapy for patients with spinal ischemic paraplegia.

Tick-borne encephalitis (TBE) vaccination: applying the most suitable vaccination schedule.

Tick-borne encephalitis (TBE) is caused by an arthropod-borne virus, belonging to the family of Flaviviridae. In case of disease, which can lead to neurological sequelae or even fatal outcomes, only symptomatic treatment is available. TBE can be prevented by vaccination. Various primary immunization schedules have been developed. To identify the most suitable schedule, the present randomised, controlled study was designed to provide data on the immune response elicited by four different immunization schedules obtained by ELISA and by neutralization test (NT). A total of 398 healthy subjects aged > or =12 years were randomised to vaccination according to either the rapid schedule (Group R, vaccination on days 0, 7 and 21), the conventional schedule (Group C, vaccination on days 0, 28 and 300), the modified conventional schedule (Group M, vaccination on days 0, 21 and 300) or the accelerated conventional schedule (Group A, vaccination on days 0, 14 and 300). Within 3 weeks (i.e. by day 21) antibody levels were higher in Group R and Group A than in Group M and Group C. Group R and Group C both had higher titres on days 42, 180 and 300, than Group A and Group M. The rapid schedule thus combines the advantages of fast protection and of high titres over the observation period of 300 days.

Experimental cauda equina compression induces HSP70 synthesis in dog.

The heat shock protein 70 (HSP70) is a key component of the stress response induced by various noxious conditions such as heat, oxygen stress, trauma and infection. In present study we have assessed the consequences of the compression of lower lumbar and sacral nerve roots caused by a multiple cauda equina constrictions (MCEC) on HSP70 immunoreactivity (HSP70-IR) in the dog. Our data indicate that constriction of central processes evokes HSP70 up-regulation in the spinal cord (L7, S1-Co3) as well as in the corresponding dorsal root ganglion cells (DRGs) (L7-S1) two days following injury. A limited number of bipolar or triangular HSP-IR neurons were found in the lateral collateral pathway (LCP) as well as in the pericentral region (lamina X) of the spinal cord. In contrast, a high number of HSP70 exhibiting motoneurons with fine processes appeared in the ventral horn (laminae VIII-IX) of lumbosacral segments. Concomitantly, close to them a few lightly HSP70-positive neuronal somata or cell bodies lacking the HSP70-IR occurred. In the DRGs, HSP70 expression was mildly up-regulated in small and medium-sized neurons and in satellite cells. On the contrary, DRGs from intact or sham-operated dogs did not reveal HSP70 specific neuronal staining. In conclusion, we have demonstrated that the MCEC in dogs mimicking the cauda equina syndrome in clinical settings evokes expression of HSP70 synthesis in specific neurons of the lumbo-sacro-coccygeal spinal cord segments and in small and medium sized neurons of corresponding DRGs. This suggests that HSP70 may play an active role in neuroprotective processes partly by maintaining intracellular protein integrity and preventing the neuronal degeneration in this experimental paradigm.

Assessing the pathogenic effect of Fusarium, Geosmithia and Ophiostoma fungi from broad-leaved trees.

Phytopathogenic effect of Geosmithia pallida, G. langdonii, Ophiostoma grandicarpum, O. querci, two isolates of O. piceae, and two isolates of Fusarium solani was compared using plant growth test (stem and root length of garden cress plants seeded on mycelium-covered potato carrot agar); Ophiostoma spp. and F. solani were isolated from oak, Geosmithia spp. from galleries of Scolytus intricatus on beech. All fungi inhibited more the root elongation than that of stems. F. solani led to plant collapse after briefly stimulating the growth of stem and in one case also root. G. langdonii inhibited stem and root growth to 20% and led to plant collapse. G. pallida inhibited root growth to 25% whereas stem growth was almost unimpaired. Ophiostoma spp. reduced stem growth to approximately 60-80% and root growth to 25-60%. O. piceae and O. querci caused plant collapse after 15-20 d.

Fluoro-Jade B evidence of induced ischemic tolerance in the rat spinal cord ischemia: physiological, neurological and histopathological consequences.

Fluoro-Jade B, a marker of degenerating neurons, was used to label histopathological changes in the rat spinal cord after transient ischemia and ischemic preconditioning (IPC). To characterize postischemic neurodegenerations and consequent neurological changes, a particular attention was paid to the standardization of ischemic conditions in animals of both groups. 1. The control ischemic rats were submitted to a reversible occlusion of descending aorta by insertion and subsequent inflation of a 2F Fogarty catheter for 12 min. 2. In the IPC rats, an episode of short 3 min occlusion and 30 min reperfusion preceded the 12 min ischemia. Postischemic motor function testing (ambulation and stepping) was provided repeatedly for evaluation of neurological status 2 h and 24 h after surgery and at the end of postischemic survival, i.e. after 48 h. Fluoro-Jade B staining was used to demonstrate degenerated neurons. In the control rats, neurological consequences of histopathological changes in lumbosacral spinal cord, manifested as paraplegia, were present after 12 min ischemia. Thus, numbers of degenerated Fluoro-Jade B positive cells were visible in gray matter of the most injured L(4)-S(2) spinal cord segments. Slight motor function impairment, consequential from significant decreasing in Fluoro-Jade B-positivity in the L(4)-S(2) spinal cord segments of the IPC rats, was considered the pathomorpfological evidence that IPC induces spinal cord tolerance to ischemia. Our results are consistent with the previously published silver impregnation method for histopathological demonstration of ischemic degeneration.

Expression of MRP2 and MDR1 transporters and other hepatic markers in rat and human liver and in WRL 68 cell line.

Here we describe a comparative study of phenotypic properties of hepatic cells in situ and in vitro. We analyzed the expression levels and distribution patterns of ABC transporters MRP2 and MDR1, pan-cytokeratin, cytokeratin 18, albumin, alpha-fetoprotein and the specific hepatocyte marker OCH1E5 in the fetal and adult rat as well as human liver tissue and in human fetal hepatocytes of WRL 68 cell line using peroxidase immunohistochemistry or immunofluorescence. Transporters MRP2 and MDR1 were expressed in all examined liver tissues, except rat ED13 embryo. The immunopositivity of these proteins was localized to the canalicular membrane of differentiating and mature hepatocytes but in the later developmental stages and in the adult liver tissues it was also found in the apical membrane of cholangiocytes. In WRL 68 cells, MRP2 and MDR1 immunoreactivity appeared after 5-6 days of cultivation and both transporters were fully expressed in the plasmalemma and in the cytoplasm 9 days after the passage. In conclusion, we observed only moderate variances reflecting diverse ontogenetic phases between the fetal and adult liver tissue. To study functions of hepatocytes in vitro, WRL 68 cells have to differentiate prior to the examination. Our findings indicate that WRL 68 cells can undergo differentiation in vitro and their antigenic profile closely resembles hepatocytes in the human liver.

Nestin expression by newly formed human blood vessels.

Nestin is a type VI intermediate filament protein originally described in neural stem cells. Here we report that immature endothelial cells generated in the course of angiogenesis express nestin. Endothelial cells of embryonic capillaries destined to vascularize growing organs also express this intermediate filament protein. Whereas nestin was sporadically expressed in mature adult human endothelial cells sporadically express nestin, this protein was consistently expressed in adult angiogenic vasculature. Nestin expression was also detected in capillaries of the corpus luteum, which replenishes itself by angiogenesis. Nestin-immunoreactive vessels were also observed in the infarcted hearts where transient ischemia triggered regeneration accompanied with neovascularization of the myocardium. Nestinpositive endothelial cells lined vessels nourishing solid growing tumors, including melanoblastomas and glioblastomas. Our data provide definitive evidence that endothelial precursors express the neural stem cell marker nestin and that this protein participates in formation of the cytoskeleton of newly formed endothelial cells. Because nestin expression was recognized under all conditions of vascular development, nestin represents a novel and reliable marker of neovascularization.

Cauda equina syndrome and nitric oxide synthase immunoreactivity in the spinal cord of the dog.

The development of the cauda equina syndrome in the dog and the involvement of spinal nitric oxide synthase immunoreactivity (NOS-IR) and catalytic nitric oxide synthase (cNOS) activity were studied in a pain model caused by multiple cauda equina constrictions. Increased NOS-IR was found two days post-constriction in neurons of the deep dorsal horn and in large, mostly bipolar neurons located in the internal basal nucleus of Cajal seen along the medial border of the dorsal horn. Concomitantly, NOS-IR was detected in small neurons close to the medioventral border of the ventral horn. High NOS-IR appeared in a dense sacral vascular body close to the Lissauer tract in S1-S3 segments. Somatic and fiber-like NOS-IR appeared at five days post-constriction in the Lissauer tract and in the lateral and medial collateral pathways arising from the Lissauer tract. Both pathways were accompanied by a dense punctate NOS immunopositive staining. Simultaneously, the internal basal nucleus of Cajal and neuropil of this nucleus exhibited high NOS-IR. A significant decrease in the number of small NOS immunoreactive somata was noted in laminae I-II of L6-S2 segments at five days post-constriction while, at the same time, the number of NOS immunoreactive neurons located in laminae VIII and IX was significantly increased. Moreover, high immunopositivity in the sacral vascular body persisted along with a highly expressed NOS-IR staining of vessels supplying the dorsal sacral gray commissure and dorsal horn in S1-S3 segments. cNOS activity, based on a radioassay of compartmentalized gray and white matter regions of lower lumbar segments and non-compartmentalized gray and white matter of S1-S3 segments, proved to be highly variable for both post-constriction periods.