Importanza prognostica delle citochine e loro ruolo nell'immunomodulazione: risultati di studi sperimentali in vivo e in vitro

Citokines are proteins produced by several cell types and secreted in response to various stimuli. These molecules are able to modify the behaviour of other cells inducing activities like growth, differentiation and apoptosis. In the last years, veterinary scientists have investigated the role played by these factors; in fact, cytokines can act as intercellular communicative signals in immune response, cell damage repair and hematopoiesis. Up to date, various cytokines have been identified and in depth comprehension of their effects in physiology, pathology and therapy is an interesting field of research. This thesis aims to understand the role played by these mediators during natural or experimentally induced pathologies. In particular, it has been evaluated the genic and protein expressions of a large number of cytokines during several diseases and starting from different matrix. Considering the heterogeneity of materials used in experimentations, multiple methods and protocols of nucleic acids and proteins extractions have been standardized. Results on cytokines expression obtained from various in vitro and in vivo experimental studies have shown how important these mediators are in regulation and modulation of the host immune response also in veterinary medicine. In particular, the analysis of inflammatory and septic markers, like cytokines, has allowed a better understanding in the pathogenesis during horse Recurrent Airway Obstruction, foal sepsis, Bovine Viral Diarrhea Virus infection and dog Parvovirus infection and the effects of these agents on the host immune system. As experimentations with mice have shown, some pathologies of the respiratory and nervous system can be reduced or even erased by blocking cytokines inflammatory production. The in vitro cytokines expression evaluation in cells which are in vivo involved in the response to exogenous (like pathogens) or endogenous (as it happens during autoimmune diseases) inflammatory stimuli could represent a model for studying citokines effects during the host immune response. This has been analyzed using lymphocytes cultured with several St. aureus strains isolated from bovine mastitic milk and different colostrum products. In the first experiment different cytokines were expressed depending on enterotoxins produced, justifying a different behaviour of the microrganism in the mammal gland. In the second one, bone marrow cells derived incubated with murine lymphocytes with colostrum products have shown various cluster of differentiation expression , different proliferation and a modified cytokines profile. A better understanding of cytokine expression mechanisms will increase the know-how on immune response activated by several pathogen agents. In particular, blocking the cytokine production, the inhibition or catalyzation of the receptor binding mechanism and the modulation of signal transduction mechanism will represent a novel therapeutic strategy in veterinary medicine.

In vivo consequences of AML1-ETO fusion protein expression for hematopoiesis

The t(8;21) (q22;q22) translocation fusing the ETO (also known as MTG8) gene on human chromosome 8 with the AML1 (also called Runx1 or CBFα) gene on chromosome 21 is one of the most common genetic aberrations found in acute myeloid leukemia (AML). This chromosomal translocation occurs in 12 % of de novo AML cases and in up to 40 % of the AML-M2 subtype of the French-American-British classification. To date, the in vivo function of aberrant AML1-ETO fusion protein expression has been investigated by several groups. However, in these studies, controversial results were reported and some key issues remain unknown. Importantly, the consequences of aberrant AML1-ETO expression for self-renewing hematopoietic stem cells (HSCs), multipotent hematopoietic progenitors (MPPs) and lineage-restricted precursors are not known. rn The aim of this thesis was to develop a novel experimental AML1-ETO in vivo model that (i) overcomes the current lack of insight into the pre-leukemic condition of t(8;21)-associated AML, (ii) clarifies the in vivo consequences of AML1-ETO for HSCs, MPPs, progenitors and more mature blood cells and (iii) generates an improved mouse model suitable for mirroring the human condition. For this purpose, a conditional tet on/off mouse model expressing the AML1-ETO fusion protein from the ROSA26 (R26) locus was generated. rn Aberrant AML1-ETO activation in compound ROSA26/tetOAML1-ETO (R26/AE) mice caused high rates of mortality, an overall disruption of hematopoietic organs and a profound alteration of hematopoiesis. However, since the generalized activity of the R26 locus did not recapitulate the leukemic condition found in human patients, it was important to restrict AML1-ETO expression to blood cell lineages. Therefore, bone marrow cells from non-induced R26/AE mice were adoptively transplanted into sublethal irradiated RAG2-/- recipient mice. First signs of phenotypical differences between AML1-ETO-expressing and control mice were observed after eight to nine months of transgene induction. AML1-ETO-expressing mice showed profound changes in hematopoietic organs accompanied by manifest extramedullary hematopoiesis. In addition, a block in early erythropoiesis, B- and T-cell maturation was observed and granulopoiesis was significantly enhanced. Most interestingly, conditional activation of AML1-ETO in chimeric mice did not increase HSCs, MPPs, common lymphoid precursors (CLPs), common myeloid progenitors (CMPs) and megakaryocyte-erythrocyte progenitors (MEPs) but promoted the selective amplification of granulocyte-macrophage progenitors (GMPs). rn The results of this thesis provide clear experimental evidence how aberrant AML1-ETO modulates the developmental properties of normal hematopoiesis and establishes for the first time that AML1-ETO does not increase HSCs, MPPs and common lineage-restricted progenitor pools but specifically amplifies GMPs. The here presented mouse model not only clarifies the role of aberrant AML1-ETO for shaping hematopoietic development but in addition has strong implications for future therapeutic strategies and will be an excellent pre-clinical tool for developing and testing new approaches to treat and eventually cure AML.rn

Identifizierung und Überexpression immunregulatorischer Moleküle in dendritischen Zellen zur gezielten Modulation ihrer T-Zell-stimulierenden Eigenschaften

Die Modifizierung von dendritischen Zellen (DCs) in vitro erfolgt meist durch eine Behandlung mit Mediatoren, die den Aktivierungszustand sowie die T-Zell-polarisierenden DC-Eigenschaften verändern. In dieser Doktorarbeit sollten zunächst mediatorinduzierte tolerogene Schlüsselmoleküle identifiziert werden. Als Modell wurden murine Knochenmarkszellen unter DC-differenzierenden Bedingungen mit dem Glucocorticoid Dexamethason (DEX) behandelt. Die generierten DEX-APCs (antigenpräsentierende Zellen) zeigten einen protolerogenen, weitgehend maturierungsresistenten Phänotyp und eine gesteigerte Expression toleranzassoziierter Moleküle. DEX-APCs induzierten in vitro aus CD25+ depletierten allogenen T-Zellen de novo CD4+ und CD8+ regulatorische T-Zellen (Tregs). Basierend auf diesen Ergebnissen und Literaturstudien wurden protolerogene Moleküle für eine Überexpression in DCs selektiert. Da DCs non-viral kaum transfizierbar sind, wurde die lentivirale Transduktion von DCs optimiert, wodurch Effizienzen bis zu 95% erreicht werden konnten. Der mit der Transduktion assoziierte physikalische Stress resultierte in einer partiellen DC-Aktivierung. Trotzdessen zeigten DCs, die die Zytokine IL-10 oder IL-21 überexprimierten, einen protolerogenen Phänotyp und induzierten in vitro Tregs. Beide DC-Populationen reduzierten im therapeutischen Ansatz im murinen Krankheitsmodell der Kontaktallergie haptenspezifisch die Ohrschwellungsreaktion. Auch DCs, die andere Zytokine, intrazelluläre Proteine oder Oberflächenrezeptoren mit protolerogenen Eigenschaften überexprimierten, wiesen eine jeweils spezifische Genexpressionssignatur auf. Diese DC-Populationen waren zumeist durch eine verminderte allogene T-Zell-Aktivierungskapazität gekennzeichnet und veränderten die Th1-/Th2-Zytokinmuster in kokultivierten T-Zellen.

Untersuchungen zu der Rolle der extrazellulären Matrix bei Fibrose und bei Tumoren

Diese Arbeit befasst sich mit der Rolle der extrazellulären Matrix und insbesondere des Proteins Fibronektin bei der Leberfibrose und bei der Einnistung von Tumorzellen in die Stammzellnische im Knochenmark.rnrnHierfür wurde in einem Fibrosemodell das Peptid pUR4b verwendet, welches die Assemblierung einer Fibronektinmatrix verhindert. Bei der Verwendung dieses Peptids während und nach der Induktion einer Leberfibrose durch die Chemikalie Dimethylnitrosamin konnte eine Verminderung der Kollagenmenge (und damit des fibrotischen Narbengewebes) in der Leber im Vergleich zu fibrotischen Kontrolltieren beobachtet werden. Darüber hinaus konnte gezeigt werden, dass dieser Effekt unabhängig von der Aktivierung der hepatischen Stellatezellen ist, jedoch zum Teil von einer verminderten Anzahl entzündlicher Zellen abhängig sein könnte. Eine verminderte Bildung von Gesamt- und aktivem TGF-β, welche zum Teil auf Effekte der verringerten Zahl der inflammatorischen Zellen zurückzuführen sein könnte, unterstützt den Effekt des verminderten Aufbaus von Narbengewebe. In vitro Untersuchungen zeigten, dass hepatische Stellatezellen bei einer Behandlung mit pUR4b weniger Fibronektin in die extrazelluläre Matrix einbauten als unbehandelte hepatische Stellatezellen. Insgesamt sprechen die Daten dafür, dass das Peptid pUR4b den Aufbau einer Fibronektinmatrix verhinderte bzw. verminderte, wodurch die Ablagerung anderer Komponenten der extrazellulären Matrix wie z.B. Kollagen gestört war und es daher zu einem Rückgang des fibrotischen Narbengewebes kam.rnrnFür die Untersuchung des Einflusses der extrazellulären Matrix und des Fibronektins bei der Einnistung von Tumorzellen wurde zunächst das Fibronektin mit Hilfe konditioneller Knockout-Mäuse in verschiedenen Zellen bzw. Organen der Tiere ausgeschaltet. Weder die Ausschaltung des zirkulierenden, noch des durch Osteoblasten und Osteozyten gebildeten, noch des zirkulierenden und von Zellen des Knochenmarks gebildeten Fibronektins beeinträchtigte die Einnistung von Tumorzellen. Auch die Bildung eines Hämatoms im Knochen hatte weder einen Einfluss auf die Einnistung von Tumorzellen noch auf die spätere Tumorentwicklung. Die Ausschaltung des tumorzellendogenen Fibronektins führte hingegen zu einer signifikant verminderten Einnistung von Tumorzellen. Diese ist wahrscheinlich auf die verstärkte Affinität dieser Tumorzellen zu Zellen des Immunsystems zurückzuführen. Diese Beobachtung konnte zum Teil durch eine verstärkte eCadherin Expression erklärt werden, welche die Bindung an verschiedene Zellen des Immunsystems vermittelt. Eine Untersuchung der osteoblastischen Stammzellnische durch die kombinierte Gabe von Parathormon und Zoledronsäure führte zu keiner Veränderung der Fibronektinkonzentration innerhalb des Knochenmarks der behandelten Tiere. Dennoch nisteten sich in dem Knochenmark der mit Parathormon und Zoledronsäure behandelten Tiere signifikant mehr Tumorzellen ein als in dem von Kontrolltieren. Dieser Effekt konnte auf einen synergetischen Effekt von Parathormon und Zoledronsäure zurückgeführt werden, der zu einer gesteigerten Osteoblastenaktivität und Änderungen der Zytokinkonzentrationen im Knochenmark führte.rnZusammenfassend zeigte sich, dass eine Veränderung der extrazelluläre Matrix und insbesondere des Proteins Fibronektin bei Leberfibrose zu einem veränderten Krankheitsbild führt und die Einnistung von Tumorzellen in das Knochenmark beeinflusst.rn

Induktion tolerogener dendritischer Zellen zur Suppression von experimenteller autoimmuner Enzephalomyelitis und Kontaktallergie

Dendritische Zellen (DC) spielen als professionelle antigenpräsentierende Zellen (APC) eine zentrale Rolle in der Aktivierung und Regulierung antigenspezifischer Immunantworten. Aus diesem Grund wird der therapeutische Einsatz von DC zur Behandlung von Autoimmunerkrankungen und Allergien sowie zur Tumorbekämpfung erforscht. Im ersten Teil der vorliegenden Arbeit untersuchten wir das Potenzial einer biolistischen DNA-Vakzinierung zur Induktion tolerogener DC in vivo. Im Tiermodell der Myelin-Oligodendrozyten-Glykoprotein Peptid 35-55 (MOGp35-55) induzierten experimentellen autoimmunen Enzephalomyelitis (EAE) sollte mittels präventiver biolistischer Kovakzinierung von Plasmid-DNA kodierend für MOG und die immunregulatorischen Zytokine TGFβ oder IL-10 eine protektive Immunität induziert werden. Die MOG-Expression stand dabei entweder unter der Kontrolle des ubiquitär aktiven CMV-Promotors oder des murinen Fascin-Promotors, um eine ektopische MOG-Expression spezifisch in dermalen DC und Langerhanszellen zu erreichen. Dass MOGp35-55-präsentierende DC nach biolistischer DNA-Vakzinierung von der Haut in die drainierenden Lymphknoten migrieren und dort T-Zellen aktivieren, konnte im Vorfeld anhand einer substanziellen Proliferation von MOGp35-55-reaktiven 2D2 T-Zellen nachgewiesen werden. Im präventiven Ansatz der MOGp35-55-induzierten EAE zeigten Mäuse, die mit MOG-kodierenden Plasmiden biolistisch transfiziert wurden, eine leicht reduzierte EAE-Symptomatik. Die Kotransfektion von MOG und TGFβ führte zu einer Verstärkung der EAE-Suppression – unabhängig davon, ob die MOG-Expression unter der Kontrolle des CMV- oder des Fascin-Promotors stand. Interessanterweise resultierte die Koapplikation von MOG- und IL-10-kodierender Plasmid-DNA nur bei DC-fokussierter MOG-Expression zu reduzierter EAE-Symptomatik. Für biolistische DNA-Vakzinierungen stellt somit der Fascin-Promotor eine potente Alternative zu viralen Promotoren dar. Entsprechend der milderen EAE-Symptome beobachteten wir bei behandelten EAE-Mäusen einen geringeren Grad an Demyelinisierung sowie eine reduzierte Infiltration des ZNS mit IFNγ-produzierenden CD4+ Th1- und IL-17-produzierenden CD4+ Th17-Zellen. Desweiteren zeigten Milzzellen ex vivo nach MOGp35-55-Restimulation eine inhibierte Proliferation und eine signifikant reduzierte IFNγ- und IL-17-Zytokinproduktion. Überraschenderweise ging die antigenspezifische Immunsuppression nicht mit der Expansion von Foxp3+ regulatorischen T-Zellen einher. Da die Milzen aber erhöhte Mengen an CD8+IFNγ+ T-Zellen aufweisen, könnte ein zytotoxisch-suppressiver Mechanismus für die Inhibition der Th1- und Th17-Immunantwort verantwortlich sein. Nachfolgende Untersuchungen sind notwendig, um die induzierten immunologischen Mechansimen mittels biolistischer DNA-Vakzinierung aufzuklären. Der zweite Teil der Arbeit befasst sich mit der Generierung von tolerogenen DC in vitro. Dafür wurden murine Knochenmarkszellen unter DC-differenzierenden Bedingungen in Gegenwart des synthetischen Glucocorticoids Dexamethason (DEX) kultiviert. Die DEX-Zugabe führte zur Differenzierung von APC mit geringer CD11c-Expression. DEX-APC waren in vitro weitestgehend gegen LPS stimulierungsresistent und zeigten eine reduzierte Expression von MHC-II und den kostimulatorischen Molekülen CD80, CD86 und CD40. Ihrem tolerogenen Phänotyp entsprechend besaßen DEX-APC ein geringeres syngenes T-Zellstimulierungspotenzial als unbehandelte BM-DC. Anhand der erhöhten Oberflächenexpression von CD11b, GR1 und F4/80 besteht eine phänotypische Ähnlichkeit zu myeloiden Suppressorzellen. Die Fähigkeit von DEX-APC in vivo antigenspezifische Toleranz zu induzieren, wurde durch einen therapeutischen Ansatz im murinen Krankheitsmodell der Kontaktallergie überprüft. Die therapeutische Applikation von DEX-APC führte hierbei im Vergleich zur Applikation von PBS oder unbehandelten BM-DC zu einer signifikant reduzierten Ohrschwellungsreaktion. Zusammenfassend demonstrieren die Ergebnisse dieser Arbeit, dass potente tolerogene DC sowohl in vivo als auch in vitro induziert werden können. Dass diese Zellpopulation effektiv antigenspezifische Immunreaktionen supprimieren kann, macht sie zu einem vielversprechenden Werkzeug in der Behandlung von Autoimmunerkrankungen und Allergien.rn

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.

Interleukin-17A stimulates granulocyte-macrophage colony-stimulating factor release by murine osteoblasts in the presence of 1,25-dihydroxyvitamin D(3) and inhibits murine osteoclast development in vitro

OBJECTIVE To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. METHODS Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte-macrophage colony-stimulating factor-knockout (GM-CSF(-/-)) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D(3)(1,25[OH](2)D(3)), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5-6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. RESULTS In both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)(2)D(3) to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF(-/-) mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. CONCLUSION These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.

Effect of monoterpenes on the formation and activation of osteoclasts in vitro

Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis. INTRODUCTION: Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied. MATERIALS AND METHODS: The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry. RESULTS: The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (> or = 1 microM) than in cultures of spleen cells (> or = 10 microM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible. CONCLUSIONS: The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

BACKGROUND: Despite a large body of clinical and histological data demonstrating beneficial effects of enamel matrix proteins (EMPs) for regenerative periodontal therapy, it is less clear how the available biological data can explain the mechanisms underlying the supportive effects of EMPs. OBJECTIVE: To analyse all available biological data of EMPs at the cellular and molecular levels that are relevant in the context of periodontal wound healing and tissue formation. METHODS: A stringent systematic approach was applied using the key words "enamel matrix proteins" OR "enamel matrix derivative" OR "emdogain" OR "amelogenin". The literature search was performed separately for epithelial cells, gingival fibroblasts, periodontal ligament cells, cementoblasts, osteogenic/chondrogenic/bone marrow cells, wound healing, and bacteria. RESULTS: A total of 103 papers met the inclusion criteria. EMPs affect many different cell types. Overall, the available data show that EMPs have effects on: (1) cell attachment, spreading, and chemotaxis; (2) cell proliferation and survival; (3) expression of transcription factors; (4) expression of growth factors, cytokines, extracellular matrix constituents, and other macromolecules; and (5) expression of molecules involved in the regulation of bone remodelling. CONCLUSION: All together, the data analysis provides strong evidence for EMPs to support wound healing and new periodontal tissue formation.

Wear particles and surface topographies are modulators of osteoclastogenesis in vitro

Prosthetic and osteosynthetic implants from metal alloys will be indispensable in orthopedic surgery, as long as tissue engineering and biodegradable bone substitutes do not lead to products that will be applied in clinical routine for the repair of bone, cartilage, and joint defects. Therefore, the elucidation of the interactions between the periprosthetic tissues and the implant remains of clinical relevance and several factors are known to affect the longevity of implants. Within this study, the effects of metal particles and surface topography on the recruitment of osteoclasts was investigated in vitro in a coculture of osteoblasts and bone marrow cells. The cells were grown in the presence of particles of different sizes and chemical composition or on metal discs with polished or sandblasted surfaces, respectively. At the end of the culture, newly formed osteoclasts were counted. Osteoclastogenesis was reduced when particles were added directly to the coculture. The effect depended on the size of the particles, small particles exerting stronger effects than larger ones. The chemical composition of the particles, however, did not affect the development of osteoclasts. In cocultures grown on sandblasted surfaces, osteoclasts developed at higher rates than they did in cultures on polished surfaces. The data demonstrate that wear particles and implant surfaces affect osteoclastogenesis and thus may be involved in the induction of local bone resorption and the formation of osteolytic lesions, leading eventually to the loosening of orthopedic implants.

Gene therapy for immunodeficiency due to adenosine deaminase deficiency

BACKGROUND: We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. METHODS: We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. RESULTS: All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). CONCLUSIONS: Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)

Progenitor cell therapies for traumatic brain injury: barriers and opportunities in translation.

Traumatic brain injury (TBI) directly affects nearly 1.5 million new patients per year in the USA, adding to the almost 6 million cases in patients who are permanently affected by the irreversible physical, cognitive and psychosocial deficits from a prior injury. Adult stem cell therapy has shown preliminary promise as an option for treatment, much of which is limited currently to supportive care. Preclinical research focused on cell therapy has grown significantly over the last decade. One of the challenges in the translation of this burgeoning field is interpretation of the promising experimental results obtained from a variety of cell types, injury models and techniques. Although these variables can become barriers to a collective understanding and to evidence-based translation, they provide crucial information that, when correctly placed, offers the opportunity for discovery. Here, we review the preclinical evidence that is currently guiding the translation of adult stem cell therapy for TBI.

Gene transfer by viral vectors

To initiate our clinical trial for chemotherapy protection, I established the retroviral vector system for human MDR1 cDNA gene transfer. The human MDR1 cDNA continued to be expressed in the transduced bone marrow cells after four cohorts of serial transplants, 17 months after the initial transduction and transplant. In addition, we used this retroviral vector pVMDR1 to transduce human bone marrow and peripheral blood CD34$\sp+$ cells on stromal monolayer in the presence of hematopoietic growth factors. These data suggest that the retroviral vector pVMDR1 could modify hematopoietic precursor cells with a capacity for long-term self renewal. Thus, it may be possible to use the MDR1 retroviruses to confer chemotherapeutic protection on human normal hematopoietic precursor cells of ovarian and breast cancer patients in whom high doses of MDR drugs may be required to control the diseases.^ Another promising vector system is recombinant adeno-associated virus (rAAV) vector. An impediment to use rAAV vectors is that production of rAAV vectors for clinical use is extremely cumbersome and labor intensive. First I set up the rAAV vector system in our laboratory and then, I focused on studies related to the production of rAAV vectors for clinical use. By using a self-inactivating retroviral vector carrying a selection marker under the control of the CMV immediate early promoter and an AAV genome with the deletion of both ITRs, I have developed either a transient or a stable method to produce rAAV vectors. These methods involve infection only and can generate high-titer rAAV vectors (up to 2 x 10$\sp5$ cfu/ml of CVL) with much less work.^ Although recombinant adenoviral vectors hardly infect early hematopoietic precursor cells lacking $\alpha\sb v\beta\sb5$ or $\alpha\sb v\beta\sb3$ integrin on their surface, but efficiently infect other cells, we can use these properties of adenoviral vectors for bone marrow purging as well as for development of new viral vectors such as pseudotyped retroviral vectors and rAAV vectors. Replacement of self-inactivating retroviral vectors by recombinant adenoviral vectors will facilitate the above strategies for production of new viral vectors. In order to accomplish these goals, I developed a new method which is much more efficient than the current methods to construct adenoviral vectors. This method involves a cosmid vector system which is utilized to construct the full-length recombinant adenoviral vectors in vitro.^ First, I developed an efficient and flexible method for in vitro construction of the full-length recombinant adenoviral vectors in the cosmid vector system by use of a three-DNA fragment ligation. Then, this system was improved by use of a two-DNA fragment ligation. The cloning capacity of recombinant adenoviral vectors constructed by this method to develop recombinant adenoviral vectors depends on the efficiency of transfection only. No homologous recombination is required for development of infectious adenoviral vectors. Thus, the efficiency of generating the recombinant adenoviral vectors by the cosmid method reported here was much higher than that by the in vitro direct ligation method or the in vivo homologous recombination method reported before. This method of the in vitro construction of recombinant adenoviral vectors in the cosmid vector system may facilitate the development of adenoviral vector for human gene therapy. (Abstract shortened by UMI.) ^