John M. Papadimitriou

Establishment of a Human Medulloblastoma Cell Line and Its Heterotransplantation into Nude Mice

A permanent cell line arising from a cerebellar medulloblastoma was established and its growth characteristics were investigated, Although the original tumor inoculum failed to take, the cultured cells were readily tumorigenic in nude mice and gave rise to rapidly growing tumors which could be easily subpassaged. The primary tumor showed evidence of both glial and neuronal differentiation, and retention of neuronal differentiation, albeit minimal, occurred in both the cultured neoplastic cells and the nude mouse tumors. Glial differentiation, on the other hand, could not be demonstrated. G-banding analysis of the chromosomes present in the cell line showed that they were exclusively human.

Influence of the epithelium on responsiveness of guinea‐pig isolated trachea to contractile and relaxant agonists

1 The potency (pD2) and maximal contractile effect (Emax) of histamine, acetylcholine, carbachol and K+ were assessed from cumulative concentration‐effect curves in guinea‐pig isolated tracheal ring preparations with and without an intact epithelium. 2 Estimates of Emax were not significantly different in epithelium‐denuded preparations compared with those measured in intact preparations; pD2 values for acetylcholine, carbachol and K+ were not significantly altered. In contrast, the potency of histamine was significantly increased by about 4 fold in preparations devoid of epithelial cells. 3 Estimates of potency and Emax were also determined for the smooth muscle relaxants isoprenaline, forskolin and theophylline (which increase intracellular cyclic AMP) and for nitroglycerin (which increases cyclic GMP) in both intact and epithelium‐stripped tracheal rings. The pD2 values for these relaxants were not significantly altered by the removal of the epithelium. However, with the exception of nitroglycerin, Emax values for these relaxants were significantly lower in stripped than in intact tracheal rings that had been maximally precontracted with carbachol. 4 The autoradiographic localisation of binding sites for the non‐selective β‐adrenoceptor ligand [125I]‐iodocyanopindolol (I‐CYP) showed that the epithelium of the guinea‐pig trachea had a 75 ± 16% greater density of β‐adrenoceptors than the smooth muscle. Removing the epithelium did not significantly alter either the density of smooth muscle binding sites or the affinity of I‐CYP binding. It was concluded that the reduced functional response of guinea‐pig trachea to isoprenaline was probably not due to smooth muscle β‐adrenoceptor dysfunction. 5 Results indicate that the epithelium plays an important role in the modulation of responsiveness of guinea‐pig trachea to histamine and relaxants that mediate their effects by selectively increasing intracellular cyclic AMP levels.

Progressive Age-Related Changes Similar to Age-Related Macular Degeneration in a Transgenic Mouse Model

Age-related macular degeneration (AMD) is the major cause of blindness in the developed world. Its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atrophy; a transgenic mouse line (mcd/mcd) expressing a mutated form of cathepsin D that is enzymatically inactive thus impairing processing of phagocytosed photoreceptor outer segments in the retinal pigment epithelial (RPE) cells. Pigmentary changes indicating RPE cell atrophy and a decreased response to flash electroretinograms were observed in 11- to 12-month-old mcd/mcd mice. Histological studies showed RPE cell proliferation, photoreceptor degeneration, shortening of photoreceptor outer segments, and accumulation of immunoreactive photoreceptor breakdown products in the RPE cells. An accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice. Transmission electron microscopy demonstrated presence of basal laminar and linear deposits that are considered to be the hallmarks of AMD. Small hard drusen associated with human age-related maculopathy were absent in the mcd/mcd mouse model at the ages analyzed. In summary, this model presents several features of AMD, thus providing a valuable tool for investigating the underlying biological processes and pathomechanism of AMD.

Macrophages: current views on their differentiation, structure, and function.

Macrophages are large mononuclear phagocytes that represent the major differentiated elements of the mononuclear phagocytic system. They arise from distinct progenitors in the bone marrow, and their immediate precursors, the monocytes, emigrate from the vascular compartment into many tissues and organs where they develop into mature macrophages. The latter display diverse morphological and functional characteristics, depending on the environmental stimuli that they receive. This phenotypic heterogeneity is, therefore, the final consequence of a series of down-regulation of some cellular processes and the up-regulation of others. The kinetics of the production of macrophages and their participation in various physiological and pathological phenomena is the subject of this review.

THE AUTOGENIC LIGAMENT PROCEDURE: A TECHNIQUE FOR PLANNED FORMATION OF AN ARTIFICIAL NEO‐LIGAMENT

Abstract A new surgical principle which can safely result in the formation of an artificial neo‐ligament, and induce the adhesion of tissues is described. Under certain conditions, an implanted Mersilene tape may act as a template, provoking a controlled linear deposition of collagen which remained unchanged, even after removal of the tape. This was proven by retropubic implantation of Mersilene tapes in female dogs, in the position of the pubo‐urethral ligament. The procedure relies on the normally negative aspects of wound healing for its effect, such as foreign body reaction, bacterial colonization, and continued tissue breakdown and regeneration.

Neutrophil depletion increases susceptibility to systemic and vaginal candidiasis in mice, and reveals differences between brain and kidney in mechanisms of host resistance

Infections caused by the yeast Candida albicans represent an increasing threat to debilitated and immunosuppressed patients, and neutropenia is an important risk factor. Monoclonal antibody depletion of neutrophils in mice was used to study the role of these cells in host resistance. Ablation of neutrophils increased susceptibility to both systemic and vaginal challenge. The fungal burden in the kidney increased threefold on day 1, and 100-fold on day 4, and infection was associated with extensive tissue destruction. However, a striking feature of the disseminated disease in neutrophil-depleted animals was the altered pattern of organ involvement. The brain, which is one of the primary target organs in normal mice, was little affected. There was a threefold increase in the number of organisms recovered from the brains of neutrophil-depleted mice on day 4 after infection, but detectable abscesses were rare. In contrast, the heart, which in normal mice shows only minor lesions, developed severe tissue damage following neutrophil depletion. Mice deficient in C5 demonstrated both qualitative and quantitative increases in the severity of infection after neutrophil depletion when compared with C5-sufficient strains. The results are interpreted as reflecting organ-specific differences in the mechanisms of host resistance.

WHAT'S NEW IN THE ROLE OF CYTOKINES ON OSTEOBLAST PROLIFERATION AND DIFFERENTIATION ?

This review assesses recent data concerning the role of cytokines produced by a variety of cells in bone on osteoblast function. The following themes are presumed: (1) osteoblasts are mesenchymal cells which act as either the major cellular agents of bone formation or as modulators of bone resorption by osteoclasts. The regulation of osteoblast proliferation and differentiation may involve a negative feedback process resulting in phenotype suppression; (2) cytokines including platelet-derived growth factors (PDGF), parathyroid hormone-related proteins (PTHrP), bone morphogenic proteins (BMP), transforming growth factor beta (TGF beta), fibroblast growth factors (FGF), insulin-like growth factors (IGF), epidermal growth factors (EGF), interleukin-1 and 6, tumour necrosis factors (TNF), interferon and haematopoietic growth factors have effects on osteoblast differentiation and proliferation but their effectiveness may not be identical in vitro and in vivo; (3) finally, therapeutic strategies for cytokine use in clinical practice are considered.

Immunocontraception Is Induced in BALB/c Mice Inoculated With Murine Cytomegalovirus Expressing Mouse Zona Pellucida 3

Abstract Immunocontraception, the prevention of oocyte fertilization through immunological means, could potentially be used to control plaguing mouse populations in Australia. This paper describes the construction of a mouse-specific betaherpesvirus, murine cytomegalovirus, which has been engineered to express the murine zona pellucida 3 (ZP3) gene. A single inoculation of this recombinant virus resulted in almost complete infertility, persistent anti-ZP3 antibody production, and profound changes to ovarian morphology in BALB/c mice in the absence of significant virus replication during the acute phase of infection. Murine cytomegalovirus may prove to be useful as a vector for the delivery of a mouse-specific immunocontraceptive agent to target populations of wild mice in the field.

Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis.

It is increasingly recognized that the morphological changes of apoptosis vary between cell types. This heterogeneity reflects the wide range of cellular proteins and enzymes involved in apoptotic pathways. Fibroblast apoptosis is crucial to the regression of scars and the restitution of healthy tissue during wound repair and may be aberrant in diseases such as idiopathic pulmonary fibrosis (IPF). The biochemical and morphological changes characterizing fibroblast apoptosis are unknown and may provide insights into the specific enzymatic mediators activated in these cells. This study aimed to examine the morphological changes of fibroblast apoptosis in both primary normal lung fibroblasts (normal‐Fb) and fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF‐Fb) and to correlate these changes with conventional biochemical markers. Transmission electron microscopy (TEM) and video time‐lapse microscopy demonstrated no difference in the duration of fibroblast apoptosis in response to FasL (6 ± 0.3 h in normal‐Fb and 6.4 ± 0.2 h in IPF‐Fb). However, IPF‐Fb were more resistant to FasL‐induced apoptosis compared with normal‐Fb. Although the majority of morphological changes of normal‐Fb and IPF‐Fb were similar, the formation of filopodia and condensation of the cytoskeletal bundles in IPF‐Fb, and more prominent vacuolation in normal‐Fb, were the significant differences between these cell subtypes. Loss of the mitochondrial membrane potential occurred prior to caspase‐3 activation, while phosphatidylserine expression, cytokeratin‐18 cleavage, and DNA fragmentation commenced after caspase‐3 activation. These observations not only suggest that specific enzymatic effectors may be preferentially activated during fibroblast apoptosis, but also provide potential insights into the pathogenesis of IPF. Copyright

Effects of Bafilomycin A1: an inhibitor of vacuolar H (+)-ATPases on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts.

Bafilomycin A1, a specific inhibitor of V‐ATPases, is a potent inhibitor of bone resorption, but the underlying mechanisms of its action remain unclear. In this study, we investigated the effect of Bafilomycin A1 on endocytosis and apoptosis in RAW cells and RAW cell‐derived osteoclasts. Quantitative analysis by flow cytometry showed that Bafilomycin A1 increased total transferrin levels when RAW cells were exposed to labeled transferrin and decreased the total uptake of Dextran‐rhodamine B, both in a dose‐ and time‐dependent fashion, indicating that Bafilomycin influences receptor‐mediated and fluid phase endocytosis in these cells. Furthermore, Bafilomycin A1 induced apoptosis of RAW cells in a dose dependent manner as evidenced by Annexin V flow cytometry. The action of Bafilomycin A1 on endocytotic events appeared to be more sensitive and occurred earlier than on its apoptosis inducing effects, suggesting that interrupting of endocytosis might be an early sign of Bafilomycin‐mediated osteoclast inhibition. Semi‐quantitative RT‐PCR analysis showed that the gene transcripts of putative Bafilomycin A1 binding subunit, V‐ATPase‐subunit a3, were expressed in the preosteoclastic RAW cell line, and up‐regulated during RANKL‐induced osteoclastogenesis. Osteoclasts treated with Bafilomycin A1 exhibited apoptosis as well as altered cellular localization of Transferrin Alexa 647. Given that endocytosis and apoptosis are important processes during osteoclastic bone resorption, the potent effect of Bafilomycin A1 on endocytosis and apoptosis of osteoclasts and their precursor cells may in part account for Bafilomycin A1 inhibited bone resorption. J. Cell. Biochem. 88: 1256–1264, 2003.