Hans Zoellner

Adult Cardiac-Resident MSC-like Stem Cells with a Proepicardial Origin

Colony-forming units - fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.

Role of Extracellular Phospholipases and Mononuclear Phagocytes in Dissemination of Cryptococcosis in a Murine Model

ABSTRACT Secreted phospholipase B (PLB) activity promotes the survival and replication of Cryptococcus neoformans in macrophages in vitro. We therefore investigated the role of mononuclear phagocytes and cryptococcal PLB in the dissemination of infection in a mouse model, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (Δplb1), and a reconstituted strain (Δplb1rec). PLB facilitated the entry of endotracheally administered cryptococci into lung IM. PLB was also required for lymphatic spread from the lung to regional lymph nodes and for entry into the blood. Langhans-type giant cells containing budding cryptococci were seen free in the lymphatic sinuses of hilar nodes of H99- and Δplb1rec-infected mice, suggesting that they may have a role in the dissemination of cryptococcal infection. The transfer of infected lung macrophages to recipient mice by tail vein injections demonstrated that these cells can facilitate hematogenous dissemination of cryptococci to the brain, independent of cryptococcal PLB secretion. PLB activities of cryptococci isolated from lung macrophages or infected brains were not persistently increased. We conclude that mononuclear phagocytes are a vehicle for cryptococcal dissemination and that PLB activity is necessary for the initiation of interstitial pulmonary infections and for dissemination from the lung via the lymphatics and blood. PLB is not, however, essential for the establishment of neurological infections when cryptococci are presented within, or after passage through, mononuclear phagocytes.

Cytokine regulation of granulocyte-macrophage colony stimulating factor and macrophage colony-stimulating factor production in human arterial smooth muscle cells

Smooth muscle cells (SMC) are the major cell type found in the walls of large blood vessels and appear to participate in local immune and inflammatory reactions, as well as in certain vascular diseases. We tested whether human arterial SMC can produce in vitro the colony stimulating factors (CSFs), granulocyte macrophage-CSF (GM-CSF) and macrophage CSF (M-CSF). Untreated internal mammary artery and aortic SMC produced no detectable GM-CSF but constitutively made M-CSF, measured by ELISA and radioimmunoassay, respectively. Interleukin-1 (IL-1) and, to a lesser extent, tumor necrosis factor alpha (TNF alpha) stimulated GM-CSF formation within 3 h; mRNA levels also increased particularly in the presence of the protein synthesis inhibitor, cycloheximide. IL-1, TNF alpha and, in addition, interferon-gamma (IFN-gamma) raised the M-CSF levels within 6 h; cycloheximide potentiated the effects of IL-1 and TNF alpha on mRNA levels. These results suggest that cytokine-stimulated human arterial SMC may be a source of the M-CSF found in atherosclerotic lesions. Since monocytes/macrophages can be activated by GM-CSF and M-CSF, while GM-CSF can also affect granulocyte function, SMC may participate in inflammatory reactions and vascular diseases by releasing these cytokines.

Marked structural and functional heterogeneity in CXCR4: Separation of HIV-1 and SDF-1α responses

CXCR4, the chemotactic cell receptor for SDF‐1α, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS‐PAGE using sucrose‐gradient‐fractionated, triton‐insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4s multiple cell functions. A comparison of wild‐type (WT) and dual N‐linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK‐293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non‐glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV‐1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF‐1α‐responsive Nalm‐6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca++ flux or a chemotactic response to SDF‐1α. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV‐1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV‐1 entry and SDF‐1α responses and may indicate therapeutic possibilities.

Nanocrystalline β-Ti alloy with high hardness, low Young's modulus and excellent in vitro biocompatibility for biomedical applications

High strength, low Youngs modulus and good biocompatibility are desirable but difficult to simultaneously achieve in metallic implant materials for load bearing applications, and these impose significant challenges in material design. Here we report that a nano-grained β-Ti alloy prepared by high-pressure torsion exhibits remarkable mechanical and biological properties. The hardness and modulus of the nano-grained Ti alloy were respectively 23% higher and 34% lower than those of its coarse-grained counterpart. Fibroblast cell attachment and proliferation were enhanced, demonstrating good in vitro biocompatibility of the nano-grained Ti alloy, consistent with demonstrated increased nano-roughness on the nano-grained Ti alloy. Results suggest that the nano-grained β-Ti alloy may have significant application as an implant material in dental and orthopedic applications.

Physiotherapeutic treatment improves oral opening in oral submucous fibrosis

BACKGROUND In oral submucous fibrosis (OSF) fibrous bands and burning mucosal pain restrict oral opening to limit speech and eating. The pathogenesis of OSF remains unclear, while surgical and pharmacological treatments have limited success, and are often inaccessible in communities using areca nut where OSF is prevalent. Improved outcomes are reported for surgical treatment when followed by physiotherapy. We tested the hypothesis that physiotherapy alone can modify tissue remodelling in OSF to increase oral opening. MATERIALS AND METHODS Fifty-four Nepali OSF patients were managed for 4 months in three randomly assigned groups receiving either: five times daily physiotherapy by inter-positioning tongue spatulas between teeth and adding a new spatula every 5-10 days; local injection of hyaluronidase with steroids; or no active treatment. RESULTS More males presented with OSF than females (p < 0.05). All patients reported reduced opening and 47% had mucosal pain. Progressive mucosal involvement was always in the same order, starting with the soft palate, and then progressing to the fauces, unilateral buccal mucosa, bilateral buccal mucosa, floor of mouth and finally lip mucosa (p < 0.006). Physiotherapy improved oral opening (p < 0.0005), but not oral pain, while no clear improvement was seen in untreated patients as well as patients managed by injection. CONCLUSIONS We conclude OSF in the Nepali population progresses in a predictable pattern, and that physiotherapy is effective for increasing the oral opening. We further suggest physiotherapy can be readily used to improve OSF in communities with otherwise limited health resources.

The Anti-Apoptotic Activity of Albumin for Endothelium Is Mediated by a Partially Cryptic Protein Domain and Reduced by Inhibitors of G-Coupled Protein and PI-3 Kinase, but Is Independent of Radical Scavenging or Bound Lipid

Increased vascular disease occurs with low albumin (human serum albumin, HSA), possibly reflecting specific inhibition of endothelial apoptosis reported for tissue culture. Despite the reported specificity for endothelial protection by HSA, the high but physiological concentrations needed appear more consistent with non-specific low-affinity interactions. We reconcile this contradiction by demonstrating protection is mediated by a partially cryptic HSA protein domain, which becomes more exposed and active following cyanogen bromide fragmentation (p < 0.001). Also, although others reported HSA radical scavenging and bound lipids as important for inhibiting apoptosis in non-endothelial cell types, we demonstrate the protective effect for endothelium is unaffected when HSA radical scavenging is blocked by alkylation, or following delipidation. Further probing the mechanism responsible, we found that the G-coupled protein inhibitors pertussis toxin and suramin reduced protection of endothelium by HSA (p < 0.005), while the tyrosine kinase inhibitor genistein had no effect. Consistent with a role for phosphoinositide 3 kinase (PI3K) was inhibition by both wortmannin and LY294002 (p < 0.05), as well as phosphorylation of Akt, while MAP kinase inhibitors had no effect. We conclude the active site in HSA inhibiting endothelial apoptosis is partially cryptic, and acts via a G-coupled protein PI3K-dependent mechanism.

Stimulation of PAI-1 expression in endothelial cells by cultured vascular smooth muscle cells.

Regulation of endothelial cell (EC) plasminogen activator inhibitor type-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (TPA) and urokinase-type plasminogen activator (UPA), by various stimuli has been well characterized. We report the upregulation of secreted and intracellular PAI-1 in human umbilical ECs when cocultured with human smooth muscle cells (SMCs) on amniotic membranes or incubated with SMC conditioned medium (CM) under serum-free conditions as determined by enzyme-linked immunosorbent assay. Cocultured human umbilical vein ECs and SMCs, or human umbilical artery ECs and SMCs, displayed a 73% and 68% increase, respectively, in released PAI-1. SMC-derived stimulatory factor release showed tissue specificity, since only human aortic, umbilical vein, and umbilical artery SMCs upregulated PAI-1 synthesis, whereas SMCs from human mammary artery, pulmonary artery, and saphenous vein did not. Stimulation of EC PAI-1 by SMC CM was both time and concentration dependent, with as much as five- and fourfold increases in supernatants and lysates, respectively. PAI-1 synthesis and activity in ECs from other vascular beds were also upregulated by SMC CM. Northern blot analysis paralleled the protein results, showing as much as a 2.7-fold increase in specific EC PAI-1 mRNA expression after incubation with SMC CM for 8 hours. PAI-1 stimulatory activity in SMC CM was completely abolished by boiling or incubation with protamine sulfate and was reduced by transient acidification or heparin-Sepharose pretreatment by 33% or 48%, respectively. The stimulatory factor(s) appeared to have a molecular mass of 23 kD as determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Dental infection and vascular disease.

Periodontitis is a chronic inflammatory response to bacterial plaque in which the anchoring bone and soft tissues supporting teeth are destroyed, resulting in tooth mobility and loss. Dental caries involves the spread of infection from the dentine to the vascular dental pulp and periapical bony tissues, before involvement of adjacent soft tissues and spreading sepsis. Several case-controlled, cross-sectional, and cohort studies report correlation between periodontitis and increased cardiovascular, cerebrovascular, and peripheral artery disease, as determined by clinical disease, angiography, ultrasonography, and reduced flow-mediated dilation. Some studies report a similar relationship of atherosclerosis with periapical infection and potentially also with coronal caries, and this review identifies the need to investigate these associations further. Smoking and cadmium exposure are epidemiologically confounding environmental risk factors shared by atherosclerosis and periodontitis. Further complicating epidemiological studies are the risk factors for both atherosclerosis and periodontitis, with which periodontitis appears to have separate positive feedback relationships. These include diabetes, increased plasma lipid levels, hypertension, and white blood cell count. Animal and human intervention studies provide some direct support of a causal role for periodontitis in atherosclerosis, and possible mechanisms include bacterial invasion of arteries, specific atherogenic properties of oral bacteria, the acute phase response, and cytokine polymorphisms.

Effect of Low Magnitude and High Frequency Mechanical Stimuli on Defects Healing in Cranial Bones

PURPOSE The aim of this investigation was to assess the effect of low magnitude high frequency (LMHF) mechanical vibrated stimulation on healing the defects surgically imposed on craniofacial bones. MATERIALS AND METHODS Forty 12-week-old C3H strain mice were separated into surgical and non-surgical groups. The surgical groups had a reproducible surgical bony lesion prepared in their right parietal bone. Both groups were further subdivided into vibration (experimental) and non-vibration (control) groups at 3 time points (zero hours, 2 weeks, and 4 weeks). The vibration groups were subjected to LMHF mechanical stimuli (30 Hz with peak strain 5 microepsilon) via a vibration machine for 20 minutes a day for 5 days a week for a total of 28 days. The specimens were analyzed using micro-computer tomography (micro-CT). RESULTS Micro-CT volumetric measurement showed that in the surgical defects groups there was a significant decrease in the volume of the healing lesion with time (P < .001) and the linear decrease was significantly more pronounced in the vibration-treated group than the nontreated group (P < .001). Micro-CT histomorphometric measurement showed that in the nonsurgical groups there was no significant difference in microstructures of bony trabeculae between vibration-treated and nontreated groups. CONCLUSION It is suggested that the introduction of LMHF mechanical stimuli in a healing bony lesion in the non-weight bearing bone significantly increases its healing capacity.