Giulio Cesare Aranzio (Arantius) (1530-89) in the pageant of anatomy and surgery

Giulio Cesare Aranzio in Italian (Julius Caesar Arantius in Latin) has not received full acclaim for his achievements in the field of anatomy and surgery that remain unknown to most physicians. His anatomical books Observationes Anatomicas, and De Humano Foetu Opusculum and surgical books De Tumoribus Secundum Locos Affectos and Hippocratis librum de vulneribus capitis commentarius brevis printed in Latin and additional existing literature on Aranzio from medical history books and journals were analysed extensively. Aranzio became Professor of Anatomy and Surgery at the University of Bologna in 1556. He established anatomy as a distinguished branch of medicine for the first time in medical history. Aranzio combined anatomy with a description of pathological processes. He discovered the 'Nodules of Aranzio' in the semilunar valves of the heart. He gave the first description of the superior levator palpebral and the coracobrachialis muscles. Aranzio wrote on surgical techniques for a wide spectrum of conditions that range from hydrocephalus, nasal polyp, goitre and tumours to phimosis, ascites, haemorrhoids, anal abscess and fistulae, and much more. Aranzio had an extensive knowledge in surgery and anatomy based in part on the ancient Greek and his contemporaries in the 16th century but essentially on his personal experience and practice.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Lung on a chip: Co-culture of alveolar epithelial cells and bone marrow derived stromal stem cells in a microfluidic system

Background: Microfluidics system are novel tools to study cell-cell interactions in vitro. This project focuses on the development of a new microfluidic device to co-culture alveolar epithelial cells and mesenchymal stem cells to study cellular interactions involved in healing the injured alveolar epithelium. Methods: Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells were seeded and injury tests were made on the cells by perfusion with media containing H2O2 or bleomycin during 6 or 18hrs. Rat Bone marrow derived stromal cells (BMSC) were then introduced into the system and cell-cell interaction was studied over 24 hrs. Results: A successful co-culture of A549 alveolar epithelial cells and BMS was achieved in the microfluidic system. The seeded alveolar epithelial cells and BMSC adhered to the bottom surface of the microfluidic device and proliferated under constant perfusion. Epithelial injury to mimic mechanisms seen in idiopathic pulmonary fibrosis was induced in the microchannels by perfusing with H2O2 or bleomycin. Migration of BMSC towards the injured epithelium was observed as well as cell-cell interaction between the two cell types was also seen. Conclusion: We demonstrate a novel microfluidic device aimed at showing interactions between different cell types on the basis of a changing microenvironment. Also we were able to confirm interaction between injured alvolar epithelium and BMSC, and showed that BMSC try to heal the injured epitelium.

The angiogenesis inhibitor vasostatin is regulated by neutrophil elastase-dependent cleavage of calreticulin in AML patients

The calcium-binding protein calreticulin (CRT) regulates protein folding in the endoplasmic reticulum (ER) and is induced in acute myeloid leukemia (AML) cells with activation of the unfolded protein response. Intracellular CRT translocation to the cell surface induces immunogenic cell death, suggesting a role in tumor suppression. In this study, we investigated CRT regulation in the serum of patients with AML. We found that CRT is not only exposed by exocytosis on the outer cell membrane after treatment with anthracyclin but also ultimately released to the serum in vitro and in AML patients during induction therapy. Leukemic cells of 113 AML patients showed increased levels of cell-surface CRT (P < .0001) and N-terminus serum CRT (P < .0001) compared with normal myeloid cells. Neutrophil elastase was identified to cleave an N-terminus CRT peptide, which was characterized as vasostatin and blocked ATRA-triggered differentiation. Levels of serum vasostatin in patients with AML inversely correlated with bone marrow vascularization, suggesting a role in antiangiogenesis. Finally, patients with increased vasostatin levels had longer relapse-free survival (P = .04) and specifically benefited from autologous transplantation (P = .006). Our data indicate that vasostatin is released from cell-surface CRT and impairs differentiation of myeloid cells and vascularization of the bone marrow microenvironment.

Interferon γ-Dependent Migration of Microglial Cells in the Retina after Systemic Cytomegalovirus Infection

Microglial cells are the resident macrophages of the central nervous system and participate in both innate and adaptive immune responses but can also lead to exacerbation of neurodegenerative pathologies after viral infections. Microglia in the outer layers of the retina and the subretinal space are thought to be involved in retinal diseases where low-grade chronic inflammation and oxidative stress play a role. This study investigated the effect of systemic infection with murine cytomegalovirus on the distribution and dynamics of retinal microglia cells. Systemic infection with murine cytomegalovirus elicited a significant increase in the number of microglia in the subretinal space and an accumulation of iris macrophages, along with morphological signs of activation. Interferon γ (IFN-γ)-deficient mice failed to induce changes in microglia distribution. Bone marrow chimera experiments confirmed that microglial cells in the subretinal space were not recruited from the circulating monocyte pool, but rather represented an accumulation of resident microglial cells from within the retina. Our results demonstrate that a systemic viral infection can lead to IFN-γ-mediated accumulation of microglia into the outer retinal layers and offer proof of concept that systemic viral infections alter the ocular microenvironment and therefore, may influence the course of diseases such as macular degeneration, diabetic retinopathy, or autoimmune uveitis, where low-grade inflammation is implicated.

Eosinophils in the gastrointestinal tract: friends or foes?

Eosinophils play an important role in the mucosal immune system of the gastrointestinal tract under resting and under inflammatory conditions. Under steady-state conditions, the mucosa of the digestive tract is the only organ harboring a substantial number of eosinophils, which, if need be, get activated and exert several effector and immunoregulatory functions. The precise function of these late-phase inflammatory cells is not yet completely understood. Nevertheless, it has recently been demonstrated that lipopolysaccharides from gram-negative bacteria activate eosinophils to rapidly release mitochondrial DNA in the extracellular space. Released mitochondrial DNA and eosinophil granule proteins form extracellular structures able to bind and inactivate bacteria. These findings suggest a novel mechanism of eosinophil-mediated innate immune responses that might be important in maintaining the intestinal barrier function. Moreover, eosinophils also play a crucial role in several inflammatory conditions, such as intestinal infections, immune-mediated inflammations and hypersensitivity reactions. Under chronic inflammatory conditions, the ability of the eosinophils to induce repair can lead to pathological sequelae in the tissue, such as esophageal remodeling in eosinophilic esophagitis. It is established that the uncontrolled eosinophilic inflammation induces fibrosis, esophageal wall thickening and strictures leading to damage that results in a loss of esophageal function. One potential mechanism of this remodeling is so-called 'epithelial mesenchymal transition', which is triggered by eosinophils and is potentially reversible under successful anti-eosinophil treatment. Therefore, eosinophils may act either as friends or as foes, depending on the microenvironment.

VEGF over-expression in skeletal muscle induces angiogenesis by intussusception rather than sprouting

Therapeutic over-expression of vascular endothelial growth factor (VEGF) can be used to treat ischemic conditions. However, VEGF can induce either normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo, which limits its clinical usefulness. The goal herein was to determine the cellular mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. We took advantage of a well-characterized cell-based platform for controlled gene expression in skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of immunodeficient mice to homogeneously over-express two specific VEGF(164) levels, previously shown to induce physiologic and therapeutic or aberrant angiogenesis, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting, but rather by intussusception, or vascular splitting. VEGF-induced endothelial proliferation without tip-cell formation caused an initial homogeneous enlargement of pre-existing microvessels, followed by the formation of intravascular transluminal pillars, hallmarks of intussusception. This was associated with increased flow and shear stress, which are potent triggers of intussusception. A similar process of enlargement without sprouting, followed by intussusception, was also induced by VEGF over-expression through a clinically relevant adenoviral gene therapy vector, without the use of transduced cells. Our findings indicate that VEGF over-expression, at doses that have been shown to induce functional benefit, induces vascular growth in skeletal muscle by intussusception rather than sprouting.

Activation of nuclear factor-kappaB in dogs with chronic enteropathies

Homeostasis in the intestinal microenvironment between the immune system and luminal antigens appears disturbed in chronic enteropathies. Pro-inflammatory cytokines likely play a role in the pathogenesis of intestinal inflammation. Several inflammatory and immunoregulatory genes have associated nuclear factor-kappaB (NF-kappaB) binding sites, which allow NF-kappaB to regulate gene transcription. The purpose of this study was to investigate (1) the occurrence of NF-kappaB activation during mucosal inflammation in situ, (2) the mucosal distribution pattern of cells expressing activated NF-kappaB within treatment groups, and (3) the effect of specific therapy on NF-kappaB activation. Dogs with chronic enteropathy were studied (n=26) and compared with 13 healthy dogs. Ten dogs had food responsive disease (FRD) and 16 had inflammatory bowel disease (IBD). NF-kappaB activation was detected in duodenal mucosal biopsies using a mouse monoclonal antibody (MAB 3026) that selectively binds the nuclear localization sequence of activated NF-kappaB. To identify macrophages, biopsies were stained using the MAC 387 antibody. Macrophages in the lamina propria double-stained for MAC 387 and NF-kappaB were quantitated; epithelial cell expression of activated NF-kappaB was determined semi-quantitatively. Results showed that more macrophages positive for activated NF-kappaB were present in lamina propria of dogs with chronic enteropathy compared to control dogs (p<0.01). More NF-kappaB positive epithelial cells were observed in FRD dogs compared to IBD dogs (p<0.05). After therapy, the number of macrophages and epithelial cells staining positive for activated NF-kappaB decreased (p<0.01) in chronic enteropathy dogs. In conclusion, activation of NF-kappaB is closely associated with the pathophysiology of canine chronic enteropathy. Down-regulation follows successful therapy.

Modulation of human osteoblasts by metal surface chemistry

The use of metal implants in dental and orthopedic surgery is continuously expanding and highly successful. While today longevity and load-bearing capacity of the implants fulfill the expectations of the patients, acceleration of osseointegration would be of particular benefit to shorten the period of convalescence. To further clarify the options to accelerate the kinetics of osseointegration, within this study, the osteogenic properties of structurally identical surfaces with different metal coatings were investigated. To assess the development and function of primary human osteoblasts on metal surfaces, cell viability, differentiation, and gene expression were determined. Titanium surfaces were used as positive, and surfaces coated with gold were used as negative controls. Little differences in the cellular parameters tested for were found when the cells were grown on titanium discs sputter coated with titanium, zirconium, niobium, tantalum, gold, and chromium. Cell number, activity of cell layer-associated alkaline phosphatase (ALP), and levels of transcripts encoding COL1A1 and BGLAP did not vary significantly in dependence of the surface chemistry. Treatment of the cell cultures with 1,25(OH)2 D3 /Dex, however, significantly increased ALP activity and BGLAP messenger RNA levels. The data demonstrate that the metal layer coated onto the titanium discs exerted little modulatory effects on cell behavior. It is suggested that the microenvironment regulated by the peri-implant tissues is more effective in regulating the tissue response than is the material of the implant itself.

Grafted neuronal precursor cells differentiate and integrate in injured hippocampus in experimental pneumococcal meningitis

Bacterial meningitis (BM) frequently causes persisting neurofunctional sequelae. Autopsy studies in patients dying from BM show characteristic apoptotic brain injury to the stem cell niche in the subgranular zone of the hippocampal dentate gyrus (DG), and this form of brain damage is associated with learning and memory deficits in experimental BM. With an eye to potential regenerative therapies, the survival, migration, and differentiation of neuronal precursor cells (NPCs) were evaluated after engraftment into the injured hippocampus in vitro and in vivo in an infant rat model of pneumococcal meningitis. Green fluorescent protein (GFP)-expressing NPCs were grafted into the DG of organotypic hippocampal slice cultures injured by challenge with live Streptococcus pneumoniae. Seven days after engraftment, NPCs had migrated from the site of injection into the injured granular layer of the DG and electro-functionally integrated into the hippocampal network. In vivo, GFP-expressing NPCs migrated within 1 week from the injection site in the hilus region to the injured granular layer of the hippocampal DG and showed neuronal differentiation at 2 and 4 weeks after transplantation. Hippocampal injury induced by BM guides grafted NPCs to the area of brain damage and provides a microenvironment for neuronal differentiation and functional integration.

An Investigation On The Use Of Microalgae For Biogas Enrichment

There is a need for biomethane capture and carbon dioxide sequestration to mitigate evident global climate change. This research work investigated the potential for microalgae to remove CO2 from biogas as a biotechnical method for upgrading the thermal value for subsequent compression, liquification, or introduction to natural gas pipelines. Because biogas is largely methane, the effect of high methane environments on mixed microalgae was explored and found that specific carbon utilization rates were not statistically different when microalgae were exposed to biogas environments (70% v/v CH4) , relative to high CO2 environment. The uses of conventional bubbled column photobioreactors (PBR) were assessed for CO2 removal and subsequent CH4 enrichment. A continuously-bubbled biogas PBR (cB-PBR5) and intermittently-bubbled biogas PBR (iB-PBR) experienced CO2 loading rates of about 1664 and 832 mg C/L*day and showed 30.0 and 60.1 % carbon removal, respectively. However, a lack of biogas enrichment and issues associated growth inhibition due to high CO2 environments as well as stripping the dissolved gases, namely oxygen and nitrogen, from the bulk liquid and introduction to the outlet gas prompted the consideration for gas/liquid separation using nonporous hollow-fiber (HF) membranes for CO2 transfer. The potential for two non-porous HF membrane materials [polydimethylsiloxane (PDMS) and composite polyurethane (PU)] were modeled along fiber length using a mechanistic model based on polymeric material transport properties (Gilmore et al., 2009). Based on a high CO2:CH4 permeability selectivity for PU of 76.2 the model predicted gas enrichment along an 8.5 cm fiber length. Because PDMS permeability selectivity is low (3.5), evident gas transfer was not predicated along a 34.3 cm length. Both of these HF materials were implemented in hollow-fiber membrane-carbonated biofilm (HFMcB) PBRs for microalgal-mediated biogas enrichment. Phototrophic biofilm colonization occurred on the membrane, where CO2 concentration was greatest. The presence of a biofilm demonstrated greater resiliency to high CO2 environments, compared to the conventional PBRs. However, as the PDMS model predicted, the PDMS HFMcBs did not demonstrate gas enrichment. These reactors received CO2 loading rates of 200 mg C/L*day based on PDMS permeability flux and showed approximately 65% removal of the total C transferred across the membrane. Thus, the HFMcBs demonstrated controlled carbonation of the bulk liquid via a nonporous HF membrane. Likewise, the experimental PU HFMcB did not show gas enrichment yet this result should be further explored due to the high permeability selectivity of the polymeric material. Chemical stratifications, namely pH and dissolved O2, present in a PDMS membrane-carbonated biofilm were analyzed using electrochemical microsensors. Results indicated that high DO (20 mg L-1) exists at surface of the biofilm where light availability is greatest and low pH microenvironments (pH=5.40) exist deep in the biofilm where the diffusive flux of CO2 drives transfer through the biofilm. The presence of a 400-600 ¿m liquid phase boundary layer was evident from microsensor profiles. Cryosectioning of the biofilm samples showed the biofilm to be approximately 1.17 ± 0.07 mm thick, suggesting that the high localized concentration of biomass associated with the phototrophic biofilm aided in overcoming inhibition in a microenvironment dominated by CO2(aq). Challenges of biofilm detachment and PBR fouling as well as microalgal growth inhibition in the presence of high CO2 content remain for applications of microalgae for biogas enrichment.

Connective tissue growth factor level is increased in patients with liver cirrhosis but is not associated with complications or extent of liver injury

Connective tissue growth factor (CTGF) is a profibrotic protein whose systemic levels are increased in liver cirrhosis. Here, association of CTGF with stages of liver injury and complications of cirrhotic liver disease has been analyzed in patients with different aetiologies of hepatic injury. CTGF is significantly increased in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 46 patients with liver cirrhosis compared to eight liver-healthy controls. In patients´ blood samples CTGF in HVS is about 6% higher than PVS levels indicating that CTGF produced in the liver is released to the circulation. CTGF is not associated with stages of liver cirrhosis defined by CHILD-PUGH or MELD score nor with secondary complications of portal hypertension (varices, ascites, spontaneous bacterial peritonitis). Transforming growth factor β (TGFβ) induces CTGF synthesis in hepatocytes and a positive association of systemic TGFβ1 and SVS and HVS CTGF is found. Three months after placing transjugular intrahepatic portosystemic shunt (TIPS) hepatic venous pressure gradient is reduced whereas CHILD-PUGH score, TGFβ1 and CTGF are not altered in serum of 15 patients. Current data show that the cirrhotic liver releases little CTGF but SVS, HVS and PVS CTGF levels are not associated with residual liver function and complications of cirrhosis.

[Spontaneous bacterial peritonitis]

Spontaneous bacterial peritonitis (SBP) is the most frequent infection in patients with cirrhosis during hospitalization and is associated with high acute and long-term mortality. Diagnosis is made by paracentesis with determination of neutrophil count in ascitic fluid. Empirical antibiotic therapy must be initiated immediately. The choice of drug is dependent on prior therapies. Liver transplantation has to be considered in the absence of contra-indications. Prophylaxis of SBP is indicated in patients with ascites and gastrointestinal hemorrhage, and in patients after SBP. Primary prophylaxis should be considered in high-risk patients with cirrhosis and ascites. The development of resistance to antibiotic drugs is a relevant side-effect.

Regulation of immune cell entry into the central nervous system

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS to not disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses can be mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly controlling immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier, which protect the CNS from the constantly changing milieu within the bloodstream, also strictly control immune cell entry into the CNS. Under physiological conditions, immune cell migration into the CNS is kept at a very low level. In contrast, during a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis, immunocompetent cells readily traverse the BBB and likely also the choroid plexus and subsequently enter the CNS parenchyma or CSF spaces. This chapter summarizes our current knowledge of immune cell entry across the blood CNS barriers. A large body of the currently available information on immune cell entry into the CNS has been derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Therefore, most of this chapter discussing immune cell entry during CNS pathogenesis refers to observations in the EAE model, allowing for the possibility that other mechanisms of immune cell entry into the CNS might apply under different pathological conditions such as bacterial meningitis or stroke.

Differential gene and protein expression in abluminal sprouting and intraluminal splitting forms of angiogenesis

In adult skeletal muscle, abluminal sprouting or longitudinal splitting of capillaries can be initiated separately by muscle overload and elevated microcirculation shear stress respectively. In the present study, gene and protein expression patterns associated with the different forms of angiogenesis were examined using a targeted gene array (Superarray), validated by quantitative RT (reverse transcription)-PCR and immunoblots. Sprouting angiogenesis induced large changes in expression levels in genes associated with extracellular matrix remodelling, such as MMP-2 (matrix metalloproteinase-2), TIMP (tissue inhibitor of metalloproteinases), SPARC (secreted protein, acidic and rich in cysteine) and thrombospondin. Changes in neuropilin, midkine and restin levels, which may underpin changes in endothelial morphology, were seen during splitting angiogenesis. Up-regulation of VEGF (vascular endothelial growth factor), Flk-1, angiopoietin-2 and PECAM-1 (platelet/endothelial cell adhesion molecule-1) was seen in both forms of angiogenesis, representing a common angiogenic response of endothelial cells. In conclusion, the present study demonstrates that general angiogenic signals from growth factors can be influenced by the local microenvironment resulting in differing forms of capillary growth to produce a co-ordinated expansion of the vascular bed.