Effects of Timing of Extracorporeal Shock Wave Therapy on Mandibular Distraction Osteogenesis: An Experimental Study in a Rat Model.

PURPOSE: Distraction osteogenesis (DO) is an established method for bone lengthening in the craniofacial skeleton. Its major drawback is the long consolidation period with attendant morbidity and possible complications. Several methods have been suggested to shorten the consolidation period. We evaluated the timing and effects of extracorporeal shock wave therapy (ESWT) on bone mineralization and extracellular bone matrix proteins during mandibular DO. MATERIALS AND METHODS: Twenty-seven rats underwent mandibular DO (latency period, 3 days; distraction period, 10 days; 0.5 mm/day) and were divided into 3 groups according to the timing of ESWT application: group I (control) received no treatment, whereas groups II and III received ESWT (0.18 mJ/mm2) before and after the active distraction period, respectively. The distracted mandibles were harvested after 4 weeks of consolidation and analyzed radiographically, histologically, and immunohistochemically. RESULTS: Group III showed significantly increased mineral density, enhanced bone formation, a higher collagen orientation index, and greater expression of type I collagen and osteocalcin proteins. CONCLUSIONS: Application of ESWT after active distraction enhances bone maturation and mineralization.

GLUT4 in murine bone growth: from uptake and translocation to proliferation and differentiation.

Skeletal growth, taking place in the cartilaginous growth plates of long bones, consumes high levels of glucose for both metabolic and anabolic purposes. We previously showed that Glut4 is present in growing bone and is decreased in diabetes. In the present study, we examined the hypothesis that in bone, GLUT4 gene expression and function are regulated via the IGF-I receptor (IGF-IR) and that Glut4 plays an important role in bone growth. Insulin and IGF-I actions on skeletal growth and glucose uptake were determined using mandibular condyle (MC) organ cultures and MC-derived primary cell cultures (MCDC). Chondrogenesis was determined by following proliferation and differentiation activities using immunohistochemical (IHC) analysis of proliferating cell nuclear antigen and type II collagen expression, respectively. Overall condylar growth was assessed morphometrically. GLUT4 mRNA and protein levels were determined using in situ hybridization and IHC, respectively. Glut4 translocation to the cell membrane was assessed using confocal microscopy analysis of GFP-Glut4 fusion-transfected cells and immunogold and electron microscopy on MC sections; glucose uptake was assayed by 2-deoxyglucose (2-DOG) uptake. Both IGF-I and insulin-stimulated glucose uptake in MCDC, with IGF-I being tenfold more potent than insulin. Blockage of IGF-IR abrogated both IGF-I- and insulin-induced chondrogenesis and glucose metabolism. IGF-I, but not insulin, induced Glut4 translocation to the plasma membrane. Additionally, insulin induced both GLUT4 and IGF-IR gene expression and improved condylar growth in insulin receptor knockout mice-derived MC. Moreover, silencing of GLUT4 gene in MCDC culture abolished both IGF-I-induced glucose uptake and chondrocytic proliferation and differentiation. In growing bone, the IGF-IR pathway stimulates Glut4 translocation and enhances glucose uptake. Moreover, intact Glut4 cellular levels and translocation machinery are essential for early skeletal growth.

TVP1022 and propargylamine protect neonatal rat ventricular myocytes against doxorubicin-induced and serum starvation-induced cardiotoxicity.

We recently reported that propargylamine derivatives such as rasagiline (Azilect) and its S-isomer TVP1022 are neuroprotective. The aim of this study was to test the hypothesis that the neuroprotective agents TVP1022 and propargylamine (the active moiety of propargylamine derivatives) are also cardioprotective. We specifically investigated the protective efficacy of TVP1022 and propargylamine in neonatal rat ventricular myocytes (NRVM) against apoptosis induced by the anthracycline chemotherapeutic agent doxorubicin and by serum starvation. We demonstrated that pretreatment of NRVM cultures with TVP1022 or propargylamine attenuated doxorubicin-induced and serum starvation-induced apoptosis, inhibited the increase in cleaved caspase 3 levels, and reversed the decline in Bcl-2/Bax ratio. These cytoprotective effects were shown to reside in the propargylamine moiety. Finally, we showed that TVP1022 neither caused proliferation of the human cancer cell lines HeLa and MDA-231 nor interfered with the anti-cancer efficacy of doxorubicin. These results suggest that TVP1022 should be considered as a novel cardioprotective agent against ischemic insults and against anthracycline cardiotoxicity and can be coadministered with doxorubicin in the treatment of human malignancies.

Effects of Extracorporeal Shock Wave Therapy on Distraction Osteogenesis in Rat Mandible.

BACKGROUND: Distraction osteogenesis has widespread clinical use in the treatment of congenital and acquired craniofacial deformities. Nonetheless, during the prolonged consolidation period, the newly regenerated bone carries the risk of complications. A known method for enhancing bone healing is extracorporeal shock wave therapy, which has been shown to induce neovascularization and promote tissue regeneration. The authors investigated whether extracorporeal shock wave therapy can accelerate bony consolidation and regeneration in distraction osteogenesis of the rat mandible and at which stage of distraction osteogenesis it should be applied. METHODS: Twenty-four male Sprague-Dawley rats were subjected to distraction osteogenesis of the right mandible (latency period, 3 days; distraction period, 10 days; 0.5 mm/day). Experimental groups consisted of the following: group I (control), no extracorporeal shock wave therapy; group II, extracorporeal shock wave therapy (0.18 mJ/mm(2)) at the latency period; and group III, extracorporeal shock wave therapy (0.18 mJ/mm(2)) at the consolidation period. Explants were removed for evaluation after 4 weeks of consolidation. RESULTS: Histologic evaluation showed well-developed cortical cortex and a higher degree of bone formation and mature bone in group III; micro-computed tomography showed significantly increased bone mineral density, bone volume fraction, and trabecular thickness; immunohistochemistry demonstrated significantly increased expression of bone morphogenetic protein-2, vascular endothelial growth factor, and proliferating cell nuclear antigen. CONCLUSION: Extracorporeal shock wave therapy application at the consolidation period during distraction osteogenesis in the rat mandible enhances bone formation and osteogenic and angiogenic growth factors, improves bone mechanical properties, and accelerates bone mineralization.

Chronic acidosis-induced growth retardation is mediated by proton-induced expression of Gs protein.

UNLABELLED: The etiology of skeletal growth retardation accompanying metabolic acidosis is not clear. Using ex vivo models for endochondral ossification, we showed that the cAMP/PKA pathway, probably triggered by proton sensitive G-protein-coupled receptors, is responsible for impaired skeletal growth in acidosis. INTRODUCTION: Chronic metabolic acidosis (CMA) is very often accompanied by skeletal growth retardation. We have previously shown in an ex vivo model of endochondral ossification that murine mandibular condyles subjected to acidic conditions exhibit growth retardation accompanied by a decline of insulin-like growth factor-I (IGF-I) and its receptors. PTH-induced ameliorative effects on the CMA-induced growth retardation of the mandibular condyle are partially mediated by protein kinase C (PKC). In this study we explored the mechanisms underlying the acidosis-induced growth retardation; in particular, the involvement of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) cellular pathway in the process. MATERIALS AND METHODS: Mandibular condyles from neonatal mice or mandibular condyle derived chondrocytes (MCDCs) were incubated for 3 days under either control or acidic conditions or in the presence of cAMP-regulating factors (cAMPrf) such as forskolin, iso-butyl methyl xanthine (IBMX), or 8-Br cAMP. The effects on proliferation and differentiation of the cultures as well as on phosphorylation of cAMP responsive element binding protein (CREB) and increased expression of the alpha subunit, Gs were determined. The intracellular pH was detected using the acridine orange assay. RESULTS: Our results show that, under acidic conditions, PKA levels were increased. H89 abolished the adverse effects of acidosis on condylar development and restored IGF-I and IGF-I receptors (IGF-IR) levels. The inhibitory effects of acidosis on proliferation and differentiation of cartilaginous cells were mimicked by cAMPrf. We have also shown that acidosis stimulates activation of Gs trimeric protein and CREB phosphorylation. GDPbetaS--a Gs antagonist--abolished the acidosis-induced condylar growth arrest. Using an acridine orange assay, we showed that the intracellular environment is not acidified under acidic conditions. CONCLUSIONS: Our results indicate that the adverse effects of acidosis on skeletal growth centers are mediated at least in part by the cAMP/PKA cellular pathway. We speculate that high proton concentrations exerted by acidosis conditions stimulate proton sensitive G-protein-coupled receptors, which are mediated by the cellular cAMP/PKA pathway and induce skeletal growth retardation.

CB1 cannabinoid receptors mediate endochondral skeletal growth attenuation by Δ9-tetrahydrocannabinol.

The endocannabinoid (EC) system regulates bone mass. Because cannabis use during pregnancy results in stature shorter than normal, we examined the role of the EC system in skeletal elongation. We show that CB1 and CB2 cannabinoid receptors are expressed specifically in hypertrophic chondrocytes of the epiphyseal growth cartilage (EGC), which drives vertebrate growth. These cells also express diacylglycerol lipases, critical biosynthetic enzymes of the main EC, and 2-arachidonoylglycerol (2-AG), which is present at significant levels in the EGC. Femora of CB1- and/or CB2-deficient mice at the end of the rapid growth phase are longer compared to wild-type (WT) animals. We find that Δ(9) -tetrahydrocannabinol (THC) slows skeletal elongation of female WT and CB2-, but not CB1-, deficient mice, which is reflected in femoral and lumbar vertebral body length. This in turn results in lower body weight, but unaltered fat content. THC inhibits EGC chondrocyte hypertrophy in ex vivo cultures and reduces the hypertrophic cell zone thickness of CB1-, but not CB2-, deficient mice. These results demonstrate a local growth-restraining EC system in the EGC. The relevance of the present findings to humans remains to be studied.

Leptin acts as a growth factor on the chondrocytes of skeletal growth centers.

Childhood obesity frequently is associated with an increase in height velocity and acceleration of epiphyseal growth plate maturation despite low levels of serum growth hormone (GH). In addition, obesity is associated with higher circulating levels of leptin, a 16-kDa protein that is secreted from the adipocytes. In this study, we evaluated the direct effect of leptin on the chondrocyte population of the skeletal growth centers in the mouse mandibular condyle, a model of endochondral ossification. We found that chondrocytes in the growth centers contain specific binding sites for leptin. Leptin, at a concentration of 0.5-1.0 microg/ml, stimulated in a dose-dependent manner the width of the chondroprogenitor zone (up to 64%), whereas higher concentrations had an inhibitory effect. Leptin induction of both proliferation and differentiation activities in the mandibular condyle was confirmed by our findings of an increase in bromodeoxyuridine (BrdU) incorporation into DNA and in (acidic) Alcian blue (AB) staining of the cartilaginous matrix. Leptin also increased the abundance of the insulin-like growth factor (IGF) I receptor and IGF-I receptor messenger RNA (mRNA) within the chondrocytes and the progenitor cell population. Our results indicate that leptin acts as a skeletal growth factor with a direct peripheral effect on skeletal growth centers. Some of its effects on the growing bone may be mediated by the IGF system via regulation of IGF-I receptor expression. We speculate that the high circulating levels of leptin in obese children might contribute to their growth.

PAX3/forkhead homolog in rhabdomyosarcoma oncoprotein activates glucose transporter 4 gene expression in vivo and in vitro.

Increased levels of glucose uptake and increased expression of the glucose transporter (GLUT) genes are characteristic features of tumors. In the muscle-derived tumor alveolar rhabdomyosarcoma (ARMS), a chromosomal translocation t(2:13) generates the PAX3/forkhead homolog in rhabdomyosarcoma (FKHR) oncoprotein. In muscle tissues, glucose transport is primarily mediated by GLUT4. However, the mechanisms that regulate GLUT4 gene expression in tumor tissues are largely unknown. Therefore, we evaluated the role of PAX3/FKHR in the regulation of GLUT4 gene expression in muscle tumorigenesis. GLUT4 mRNA and protein were detected in ARMS-derived human biopsies and in ARMS-derived RH30 myoblasts, which both express the PAX3/FKHR chimeric protein, but not in either C2C12 or embryonal rhabdomyosarcoma-derived myoblasts. GLUT4 was functionally active in RH30 cells, because insulin induced a 1.4-fold stimulation of basal 2-deoxyglucose uptake rates. Coexpression of PAX3/FKHR increased basal transcriptional activity from a GLUT4 promoter reporter (GLUT4-P) in C2C12, SaOS-2, and Chinese hamster ovary-K1 cells in a dose-dependent and tissue-specific manner. PAX3/FKHR mutants with deletions in either the homeodomain (DeltaHD) or the FKHR-derived activation domain (DeltaFKHR), or in which the PAX3-derived paired domain (PD) was point-mutated (PD-R56L), were unable to activate GLUT4-P. Progressive 5'-deletion analysis of GLUT4-P further identified a specific region of the promoter, -66/+163 bp, which retained about 65% of the full transactivation effect. EMSA studies established that the PAX3/FKHR protein directly and specifically binds to this region and to a shorter fragment, -4/+36 bp, that contains potential binding sites for HD and PD, but not to a -4/+36-bp fragment whose HD and PD sites have been mutated. Thus, the functional interaction of PAX3/FKHR with GLUT4-P appears to require all of the functional domains of PAX3/FKHR, as well as a -4/+36-bp region within the GLUT4 promoter. Taken together, the data suggest that the GLUT4 gene is a downstream target of PAX3/FKHR and that GLUT4 is aberrantly transactivated by this oncoprotein both in vivo and in vitro.

Attenuation of 6-hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-kappaB) activation and cell death by tea extracts in neuronal cultures.

Antioxidant and anti-inflammatory therapy approaches have been in the focus of attention in the treatment of neurodegenerative Parkinson's and Alzheimer's diseases where oxidative stress has been implicated. Tea extracts have been previously reported to possess radical scavenger, iron chelating and anti-inflammatory properties in a variety of tissues. The purpose of this study was to investigate potential neuroprotective effects of tea extracts and possible signal pathway involved in a neuronal cell model of Parkinson's disease. We demonstrated highly potent antioxidant-radical scavenging activities of green tea (GT) and black tea (BT) extracts on brain mitochondrial membrane fraction, against iron (2.5 microM)-induced lipid peroxidation. Both extracts (0.6-3 microM total polyphenols) were shown to attenuate the neurotoxic action of 6-hydroxydopamine (6-OHDA)-induced neuronal death. 6-OHDA (350 and 50 microM) activated the iron dependent inflammatory redox sensitive nuclear factor-kappaB (NF-kappaB) in rat pheochromocytoma (PC12) and human neuroblastoma (NB) SH-SY5Y cells, respectively. Immunofluorescence and electromobility shift assays showed increased nuclear translocation and binding activity of NF-kappaB after exposure to 6-OHDA in NB SH-SY5Y cells, with a concomitant disappearance from the cytoplasm. Introduction of GT extract (0.6, 3 microM total polyphenols) before 6-OHDA inhibited both NF-kappaB nuclear translocation and binding activity induced by this toxin in NB SH-SY5Y cells. Neuroprotection was attributed to the potent antioxidant and iron chelating actions of the polyphenolic constituents of tea extracts, preventing nuclear translocation and activation of cell death promoting NF-kappaB. Brain penetrating property of polyphenols may make such compounds an important class of drugs for treatment of neurodegenerative diseases.

Iron and alpha-synuclein in the substantia nigra of MPTP-treated mice: effect of neuroprotective drugs R-apomorphine and green tea polyphenol (-)-epigallocatechin-3-gallate.

One of the prominent pathological features of Parkinson's disease (PD) is the abnormal accumulation of iron in the substantia nigra pars compacta (SNpc), in the reactive microglia, and in association with neuromelanin, within the melanin-containing dopamine (DA) neurons. Lewy body, the morphological hallmark of PD, is composed of lipids, redox-active iron, and aggregated alpha-synuclein, concentrating in its peripheral halo and ubiquitinated, hyperphosphorylated, neurofilament proteins. The capacity of free iron to enhance and promote the generation of toxic reactive oxygen radicals has been discussed numerous times. Recent observations, that iron induces aggregation of inert alpha-synuclein to toxic aggregates, have reinforced the critical role of iron in oxidative stress-induced pathogenesis of DA neuron degeneration and protein degradation via ubiquitination. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6-hydroxydopamine-induced neurodegeneration in rodents and nonhuman primates is associated with increased presence of iron and alpha-synuclein in the SNpc. The accumulation of iron in MPTP-induced neurodegeneration has been linked to nitric oxide-dependent mechanism, resulting in degradation of prominent iron regulatory proteins by ubiquitination. Radical scavengers such as R-apomorphine and green tea catechin polyphenol (-)-epigallocatechin-3-gallate, as well as the recently developed brain-permeable VK-28 series derivative iron chelators, which are neuroprotective against these neurotoxins in mice and rats, prevent the accumulation of iron and alpha-synuclein in SNpc. This study supports the notion that a combination of iron chelation and antioxidant therapy, as emphasized on several occasions, might be a significant approach to neuroprotection in PD and other neurodegenerative diseases.

Absence of intraepidermal glycosyltransferase ppGalNac-T3 expression in familial tumoral calcinosis.

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare autosomal recessive disorder characterized by progressive, tumor-like calcifications in the dermis and subcutaneous tissues. The disease is associated with primary hyperphosphatemia due to increased renal tubular reabsorption of phosphate. We recently identified mutations in GALNT3 as the proximal cause of this metabolic disorder. GALNT3 encodes the glycosyltransferase UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 3 (ppGalNAc-T3), which initiates mucin-type O-glycosylation and thus takes part in posttranslational modification and formation of mucin-type glycoproteins. A number of studies have previously described the histopathological and ultrastructural features of lesional skin in HFTC, but little is currently known about the morphology of the normal-appearing non-lesional skin. We obtained biopsies of uninvolved skin from two HFTC patients carrying a known splice site mutation in GALNT3. Light and electron microscopic examination of a biopsy of one of the two patients did not reveal abnormal findings in the epidermis or dermis. However, immunohistochemical studies of frozen skin sections of biopsies of the two patients using monoclonal antibodies directed against three ppGalNac isoforms revealed the complete absence of immunostaining for ppGalNAc-T3 while the staining pattern for ppGalNAc-T2 and -T6 was identical in skin biopsies obtained from HFTC patients and healthy control individuals. Our data provide for the first time evidence for ppGalNAc-T3 deficiency in the skin of HFTC patients and suggest that immunostaining of skin biopsy samples for ppGal-Nac-T3 might be a useful tool for the diagnosis of HFTC.

PTH ameliorates acidosis-induced adverse effects in skeletal growth centers: the PTH-IGF-I axis.

BACKGROUND: Chronic metabolic acidosis (CMA) exerts profound adverse effects on bone metabolism thereby leading to impaired skeletal linear growth. We have recently shown that CMA in vitro causes distinct morphological changes in skeletal growth centers along with inhibition of endochondral differentiation. In addition, CMA causes an end organ resistance to the anabolic effects of growth hormone (GH) and locally produced insulin-like growth factor-I (IGF-I) in skeletal growth centers. Given the effects of parathyroid hormone (PTH) and PTH related protein (PTHrP) on the development of cartilaginous bone, we sought to determine whether PTH has any effects on the changes induced by CMA in skeletal growth centers. The interaction between PTH and IGF-I in growth centers during neutral or acidic conditions were studied specifically. METHODS: An in vitro organ culture system using the murine mandibular condyle was employed as a model for endochondral active growth center. Condyles from six-day-old mice were cultured in BGJb medium of either neutral pH (pH approximately 7.4) or acidic pH (pH approximately 7.15) in the presence or absence of 10-10 mol/L [1-34] PTH. After 24, 48, 72 and 96 hours of culture, the condyles were washed, fixed in formaldehyde, and processed for paraffin embedding. Histologic markers of the growth center were assessed. In addition, the protein level and mRNA expression for various markers of cartilage differentiation were evaluated by immunohistochemistry and in situ hybridization, respectively. The abundance and expression levels of IGF-I and IGF-I receptor (IGF-I-R) were assessed also. RESULTS: Following incubation for 72 hours in acidic conditions, there was a marked attenuation of the chondroblastic zone, suggesting a defect in the process of cellular differentiation. Acidosis also down-regulated endochondral differentiation markers (cartilage specific proteoglycans, collagen type II). This was accompanied by a reduction in the expression of IGF-1, IGF-1 receptor and PTH receptors. PTH (10-10 mol/L) added to acidic cultures prevented the adverse effects of CMA on endochondral differentiation and increased the overall condylar growth, when compared to acidic conditions without PTH. PTH also up-regulated its own receptor in control as well as during acidic conditions, and increased the expression levels of IGF-1 and IGF-1 receptor in the acidotic condyle. Acidosis increased the expression of IGF-I binding protein-4 (IGFBP-4, an inhibitor of IGF-I activity), whereas coincubation with PTH during acidic conditions abrogated the up-regulation of IGFBP-4. Addition of a neutralizing antibody to IGF-I-R during PTH treatment under acidic conditions resulted in the abrogation of the ameliorative effect of PTH on endochondral differentiation. The protein kinase C (PKC) signaling pathway was modulated negatively by CMA. However, PTH activated PKC-alpha under both control and acidic conditions. The phorbol ester, PMA (phorbol 12-myristate 13-acetate), a PKC activator, mimicked the effect of PTH on chondrocyte differentiation. CONCLUSION: Parathyroid hormone at low concentration stimulates the differentiation and proliferation of cartilage cells and prevents the suppressive effect of acidosis on endochondral bone differentiation and on the IGF-I/IGF-I-R system in skeletal growth centers. Increased local production of IGF-I by PTH, which takes place even during acidotic conditions, mediates, at least in part, the ameliorative effect of PTH. Protein kinase C is probably one of the signaling pathways mediating the salutary effects of PTH on chondrocyte differentiation in growth centers. This study lends further credence to the notion that under certain conditions, PTH or PTHrP can exert anabolic effects in the skeleton. These findings may be of clinical-therapeutic significance in children and patients with CMA.

Early and late gene changes in MPTP mice model of Parkinson's disease employing cDNA microarray.

Recently, we reported specific brain gene expression changes in the chronic MPTP model inthe late stage of degeneration, employing cDNA expression array, which indicate a "domino" cascade of events involved in neuronal cell death. In an attempt to elucidate early gene expression profile in the region of the substantia nigra (SN) and the striatum of acute MPTP-treated mice (3-24 h), we elected a restricted number of genes affected by the long-term MPTP treatment, and their expression was examined. Specifically, we detected alterations in the expression of genes implicated in oxidative-stress, inflammatory processes, signal transduction and glutamate toxicity. These pro-toxic genes appear to be compensated by the elevated expression in trophic factors and antioxidant defenses, which are also activated by short exposure to MPTP. The time course of these gene expression changes indicates the importance of investigating the early gene cascade of events occurring prior to late nigrostriatal dopamine neuronal cell death.

Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

IL-27 is a recently defined family member of the long-chain, four-helix bundle cytokines, which consist of EBI3, an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide. The role of IL-27 in the regulation of experimental autoimmune encephalomyelitis has never been studied. We show in this study that neutralizing the in vivo function of IL-27 by Abs against IL-27 p28 rapidly suppressed an ongoing long-lasting disease in C57BL/6 mice. These Abs were then used to determine the mechanistic basis of disease suppression. We show in this study that IL-27 is involved not only in the polarization of naive T cells undergoing Ag-specific T cell activation, but also in promoting the proliferation and IFN-gamma production by polarized T cells, including the long term Th1 line that has been previously selected against the target encephalitogenic determinant. This may explain in part why neutralizing IL-27 suppresses an already established disease in a very rapid and significant manner.

Targeting the function of IFN-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis.

IFN-gamma-inducible protein 10 (IP-10) is a CXC chemokine that is thought to manifest a proinflammatory role because it stimulates the directional migration of activated T cells, particularly Th1 cells. It is an open question whether this chemokine is also directly involved in T cell polarization. We show here that during the course of adjuvant-induced arthritis the immune system mounts a notable Ab titer against self-IP-10. Upon the administration of naked DNA encoding IP-10, this titer rapidly accelerates to provide protective immunity. Self-specific Ab to IP-10 developed in protected animals, as well as neutralizing Ab to IP-10 that we have generated in rabbits, could inhibit leukocyte migration, alter the in vivo and in vitro Th1/Th2 balance toward low IFN-gamma, low TNF-alpha, high IL-4-producing T cells, and adoptively transfer disease suppression. This not only demonstrates the pivotal role of this chemokine in T cell polarization during experimentally induced arthritis but also suggests a practical way to interfere in the regulation of disease to provide protective immunity. From the basic science perspective, this study challenges the paradigm of in vivo redundancy. After all, we did not neutralize the activity of other chemokines that bind CXCR3 (i.e., macrophage-induced gene and IFN-inducible T cell alpha chemoattractant) and yet significantly blocked not only adjuvant-induced arthritis but also the in vivo competence to mount delayed-type hypersensitivity.

Early and late molecular events in neurodegeneration and neuroprotection in Parkinson's disease MPTP model as assessed by cDNA microarray; the role of iron.

Possible cell death mechanisms for pars compacta nigro-striatal dopamine neurons in Parkinson's disease include oxidative stress, inflammatory processes, nitric oxide iron accumulation, glutamate toxicity and diminished neurotrophic factor responses. There is a notion that Parkinson's disease is not a single disorder but a syndrome that can be initiated by several factors. Because of limitations of biochemical methods in the global analysis of neuronal death, a full picture of events has not been established. However, recently developed cDNA microarray or microchips, in which the global expression of thousands of genes can be assessed simultaneously, is changing the prospect for understanding the disease process, its progression, response to drugs, etc. The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is considered the most valid model of Parkinson's disease. We employed the technique of cDNA microarray gene expression to determine the mechanism of action of MPTP in mouse substantia nigra. Also, we studied neuroprotective processes induced by several compounds, including R-apomorphine and the green tea polyphenol epigallo-catechin-3-gallate (EGCG). This was done in two ways: (1) the time-dependent acute effect of MPTP, for determining which of the initial genes might lead to dopamine neuron death and (2) gene expression at the time of MPTP-induced dopamine neuron death. We observed that early (acute MPTP) gene expression differs from effects seen at the time of death (chronic MPTP), and that early gene changes are crucial for setting into action genes that eventually cause dopamine neuron death. Furthermore, this process is a cascade of "domino" effects, some of which were previously established by biochemical means. However, our findings show an additional large number of events previously unknown. The neuroprotective drugs reversed some but not all of the gene expression, suggesting involvement of these genes in the neurodegenerative process. Because of the profound complexity of "domino" effect it is now reasonable to understand why a single neuroprotective drug has not shown clinical neuroprotective efficacy. Future multi neuroprotective drugs may be necessary for treatment of not only Parkinson's disease, but other neurodegenerative diseases (e.g. Alzheimer's disease) and detrimental states (e.g. ischaemia).

Suppression of ongoing adjuvant-induced arthritis by neutralizing the function of the p28 subunit of IL-27.

IL-27 is a recently defined family member of the long-chain four-helix bundle cytokines, which consists of EBI3, an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide. The role of IL-27 in the regulation of inflammatory autoimmune diseases has never been studied. The current study uses the DNA vaccination technology, and highly specific Abs to the p28 subunit of IL-27 that were generated by this technology, to delineate its role in the regulation of adjuvant-induced arthritis in Lewis rats. Neutralizing the in vivo function of IL-27 by targeted DNA vaccines and by Abs against IL-27 p28 that were produced in protected donors could rapidly suppress an ongoing disease. Disease suppression was associated with a reduced ex vivo production of inflammatory cytokines. We then used these Abs to investigate the mechanistic basis of disease suppression, showing that IL-27 is not only involved in directing the polarization of naive T cells, but also affects the proliferative response and cytokine production of Ag-specific effector/memory Th1 cells. This may explain, in part, its important role in the regulation of inflammatory autoimmune diseases, and also suggest novel ways of therapy.