Effect of hydroxyapatite/tricalcium-phosphate coating on osseointegration of plasma-sprayed titanium alloy implants

This study determined the effects of a plasma-sprayed hydroxyapatite/tricalcium phosphate (HA/TCP) coating on osseointegration of plasma-sprayed titanium alloy implants in a lapine, distal femoral intramedullary model. The effects of the HA/TCP coating were assessed at 1, 3, and 6 months after implant placement. The HA/TCP coating significantly increased new bone apposition onto the implant surfaces at all time points. The ceramic coating also stimulated intramedullary bone formation at the middle and distal levels of the implants. Fluorescent bone labeling indicated that new bone formation occurred primarily during the first 3 months after implantation, with comparatively little activity detected in the latter stages of the study. There was no associated increase in pullout strength at either 3 or 6 months; however, post-pullout evaluation of the implants indicated that the HA/TCP coating itself was not the primary site of construct failure. Rather, failure was most commonly observed through the periprosthetic osseous struts that bridged the medullary cavity. The demonstrated osteoconductive activity of HA/TCP coating on plasma-sprayed titanium alloy implant surfaces may have considerable clinical relevance to early host-implant interactions, by accelerating the establishment of a stable prosthesis-bone interface.     

Augmented expression of daintain/allograft inflammatory factor-1 is associated with clinical disease: dynamics of daintain/allograft inflammatory factor-1 expression in spleen, peripheral nerves and sera during experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is an animal model of Guillain-Barré syndrome, characterized by inflammation and demyelination of the peripheral nervous system (PNS). Daintain/allograft inflammatory factor-1 (daintain/AIF-1) is a novel interferon-gamma-inducible protein expressed by macrophages during organ specific autoimmune diseases. To study the involvement of daintain/AIF-1 in EAN we induced EAN in Lewis rats by immunizing with bovine PNS myelin (BPM) and complete Freund's adjuvant (CFA). The expression of daintain/AIF-1 was examined in the spleen, peripheral nerves and sera during the course of EAN by immunohistochemistry and radioimunoassay (RIA). The expression of daintain/AIF-1 in the spleen and in the sciatic nerves peaked at the preclinical stage (day 7 post immunization (p.i.)) and at the height (day 15 p.i.) of clinical EAN, consistent with a disease promoting role for daintain/AIF-1. Daintain/AIF-1 expressing cells represented a subset of ED1+ or CD11b/c+ mononuclear cells. A significant increase of daintain/AIF-1-like immunoreactivity in sera occurred at the preclinical stage of EAN. Taken together, these data indicate that daintain/AIF-1 may play a proinflammatory role in the pathogenesis of EAN.     

The effect of pretreating morselized allograft bone with rhBMP-2 and/or pamidronate on the fixation of porous Ti and HA-coated implants.

BMPs stimulate new bone formation, but may also accelerate bone resorption. We added rhBMP-2 and pamidronate to morselized bone allograft packed around uncemented HA-coated and non-coated porous Ti implants in sixteen dogs. Each dog received four implants where the allograft was added (1) nothing, (2) BMP, (3) BP, and (4) BMP+BP. After four weeks, the untreated control implants had better mechanical fixation than all other treatment groups. The rhBMP-2-treated group had abundant formation of new bone on and around the implant. However, almost all allografts were resorbed, rendering the implant mechanically unstable. In the pamidronate-treated group the allograft was preserved, but the implants were covered by fibrous tissue and there was almost no new bone formation. This was also the case for the combined BMP+BP group, although fibrous tissue was absent on these implants. The HA-coated implants had more than twice as good mechanical fixation and improved osseointegration compared to the corresponding Ti implants. RhBMP-2 raised the total metabolic turnover of bone within the allograft with a net negative result on implant fixation. Pamidronate virtually blocked bone metabolism, also when combined with rhBMP-2. The results warrant a conservative approach and emphasize the importance of identifying a therapeutic window for these substances prior to clinical use.     

Regulation of endothelial VCAM-1 expression in murine cardiac grafts. Expression of allograft endothelial VCAM-1 can be manipulated with antagonist of IFN-alpha or IL-4 and is not required for allograft rejection.

This report provides evidence to support the hypothesis that tumor necrosis factor-alpha (TNF-alpha) and IL-4 promote the expression of new endothelial surface molecules in rejecting murine heterotopic cardiac allografts. The microvascular endothelia of these cardiac allografts all develop strong reactivity with the monoclonal antibodies (mAbs) YN1.1/74 (anti-ICAM-1), M/K-2 (anti-VCAM-1) and MECA-32 (undefined molecule) within 3 to 5 days of graft implantation. Daily treatment of the allograft recipients with pentoxifylline (PTX), soluble TNF receptor (TNFR:Fc), anti-interleukin-4 (IL-4) mAb (11B11), or soluble IL-4 receptor, each abrogate the expression of endothelial VCAM-1 and reduce the endothelial reactivity with the mAbs YN1.1/74 and MECA-32 to levels found in cardiac isografts. This is accompanied by a reduction, but not an elimination, of interstitial leukocytic infiltration. Despite this, cardiac allograft recipients treated with PTX or the mAb 11B11 rejected allografts at the same rate as untreated allograft recipients, ie, within 10 to 12 days after graft implantation. These rejected grafts contained mRNAs for TNF-alpha and IL-4, as well as for all other cytokines that have been associated with rejecting allografts. This suggests that endothelial VCAM-1 expression, which is characteristic of rejection, is not an essential element of the rejection process. Interestingly, the grafts from the PTX-treated recipients continued to display rare, isolated VCAM-1 positive cells in the interstitium, which may be dendritic cells. In general, these studies demonstrate a role for IL-4 and TNF-alpha in the alterations of vascular endothelial phenotype observed in rejecting cardiac allografts. They also demonstrate that endothelial VCAM-1 expression is not essential for the allograft rejection process, and that the role of VCAM-1 in this process may be more subtle than was initially suspected.     

Effects of pranlukast, a cysteinyl leukotriene receptor 1 antagonist, combined with inhaled beclomethasone in patients with moderate or severe asthma.

BACKGROUND: Although inhaled steroids are used as the first line of therapy in asthmatic patients, symptoms of asthma do not improve completely in some patients. OBJECTIVE: To investigate the effects of pranlukast, a cysteinyl leukotriene receptor 1 antagonist, in patients with moderate/severe asthma, when combined with beclomethasone dipropionate (BDP). METHODS: Protocol 1: After a 2-week observation period, 41 patients with moderate asthma were divided into those receiving BDP at 1,600 microg/day or 800 microg/day + pranlukast (450 mg/day). The effect of treatment was evaluated by measuring AM peak expiratory flow rate, symptom score, frequency of beta2-agonists, and daily variability of peak expiratory flow rate. Protocol 2: 39 patients participated in this study including those with moderate asthma on 800 microg/day BDP (group I), severe asthma on BDP at 1,600 microg/day (group II), and severe asthma on 1,600 microg/day BDP + 5 to 20 mg prednisolone (group III). Patients of all groups were additionally treated with pranlukast. RESULTS: Protocol 1: Both treatment regimens resulted in improvement in each clinical parameter. There were no significant differences in the effects of two treatment regimens. Protocol 2: Pranlukast was effective in group I and II, but not in group III. In groups I and II, pranlukast tended to be more effective when BDP was introduced within the first year of onset of asthma. CONCLUSIONS: Pranlukast is effective for patients with moderate asthma and those patients with severe asthma who are not treated with oral steroids. Pranlukast is more effective in patients treated with BDP early after onset.     

Regulation of the atheroma-enriched protein, SPRR3, in vascular smooth muscle cells through cyclic strain is dependent on integrin alpha1beta1/collagen interaction

Atherosclerotic plaques express high levels of small proline-rich repeat protein (SPRR3), a previously characterized component of the cornified cell envelope of stratified epithelia, where it is believed to play a role in cellular adaptation to biomechanical stress. We investigated the physiological signals and underlying mechanism(s) that regulate atheroma-enriched SPRR3 expression in vascular smooth muscle cells (VSMCs). We showed that SPRR3 is expressed by VSMCs in both human and mouse atheromas. In cultured arterial VSMCs, mechanical cyclic strain, but neither shear stress nor lipid loading induced SPRR3 expression. Furthermore, this upregulation of SPRR3 expression was dependent on VSMC adherence to type I collagen. To link the mechanoregulation of SPRR3 to specific collagen/integrin interactions, we used blocking antibodies against either integrin α1 or α2 subunits and VSMCs from mice that lack specific collagen receptors. Our results showed a dependence on the α1β1 integrin for SPRR3 expression induced by cyclic strain. Furthermore, we showed that integrin α1 but not α2 subunits were expressed on VSMCs within mouse lesions but not in normal arteries. Therefore, we identified the enrichment of the mechanical strain-regulated protein SPRR3 in VSMCs of both human and mouse atherosclerotic lesions whose expression is dependent on the collagen-binding integrin α1β1 on VSMCs. These data suggest that SPRR3 may play a role in VSMC adaptation to local biomechanical stress within the plaque microenvironment.     

The latent membrane protein-1 in Epstein-Barr virus-transformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organizing centre.

Immunofluorescence studies on Epstein-Barr virus (EBV)-transformed lymphoblastoid cells have previously shown that the latent membrane transforming protein (LMP-1) is found in patch-like inclusions which also immunostain for vimentin. We now show that EBV transformation causes a major reorganization of intermediate filaments, microtubules, mitochondria, and lysosomal elements, which generally become oriented around the microtubule organizing centre. Immunogold electron microscopy shows that LMP-1 is primarily concentrated in secondary lysosomes together with ubiquitin-protein conjugates and heat-shock protein 70. Intermediate filament inclusion formation with the above characteristics may be a general response triggered by other membrane glycoproteins; as seen, for example, in major human neurodegenerative diseases such as diffuse Lewy body disease.     

Modulation of synaptosomal protein phosphorylation/dephosphorylation by calcium is antagonised by inhibition of protein phosphatases with okadaic acid.

The protein phosphatase inhibitor okadaic acid was used to investigate the protein phosphatases involved in the endogenous dephosphorylation of proteins in intact synaptosomes. Despite the fact that the calcium-dependent protein phosphatase (calcineurin) is most concentrated in synaptosomes and accounts for approximately 0.3% of synaptoplasmic protein, the majority of the dephosphorylation activity under both basal and depolarisation conditions is due to protein phosphatase type 1 (PP1) and/or protein phosphatase type 2A (PP2A). Nevertheless our results do suggest that calcineurin is active in synaptosomes and has 2 effects: a rapid, direct dephosphorylation of a limited range of substrates and an indirect activation of PP1 presumably by dephosphorylation of protein phosphatase 1 inhibitor-1.     

Hypermethylation of APC promoter 1A is associated with moderate activation of Wnt signalling pathway in a subset of colorectal serrated adenomas

Aims:  The role of Wnt signalling pathway in serrated adenomas (SAs) remains to be identified. The aim of this study was to determine whether Wnt signalling plays a role in the pathogenesis of SAs, and to clarify the mechanism of Wnt signalling activation in SAs.
Methods and results:  This study investigated immunoreactivities of adenomatous polyposis coli (APC) and β-catenin, mutations of APC and β-catenin genes, methylation status of APC promoter 1A in 12 SAs, and compared the findings with normal colorectal mucosa, hyperplastic polyps, traditional adenomas (TAs) and colorectal cancers (CRCs). APC expression was moderately decreased in SAs. Cytoplasmic accumulation of β-catenin was demonstrated in 41.7% (5/12) of SAs, but membranous immunoreactivity of β-catenin was lost in only 8.3% (1/12) of SAs. No β-catenin mutation was detected in any of 12 SAs, and only one SA was found to be positive for APC gene mutation. Complete methylation of APC promoter 1A was found in 41.7% (5/12) of SAs, but in no TAs or CRCs.
Conclusions:  Hypermethylation of APC promoter 1A, instead of mutations involving APC and β-catenin , contributes to moderate activation of Wnt signalling in a subset of SAs.     

Low expression of p27 protein combined with altered p53 and Rb/p16 expression status is associated with increased expression of cyclin A and cyclin B1 in diffuse large B-cell lymphomas.

The expression of the cyclin-dependent kinase inhibitor (CDKI) p27 protein was investigated in relation to (1) the expression of the cell cycle regulators p53, Rb and p16 and (2) the proliferation profile as determined by the expression of Ki67, cyclin A, and cyclin B1 in 80 cases of de novo diffuse large B-cell lymphomas (DLBCL). P27 expression was low/null in large tumor cells in 58/80 cases and intermediate/high in 22/80 cases. Increased expression of p53 protein was observed in 39/80 cases. Decreased expression of Rb and p16 proteins was mutually exclusive and was observed in 5/80 and 14/80 cases, respectively. The analysis of the p27 expression status (low/null versus intermediate/high) with respect to the p53 and/or Rb/p16 expression status showed that low/null p27 expression was significantly correlated with increased p53 expression (P =.018) and showed a strong trend for correlation with concurrent increased p53 expression and decreased Rb or p16 expression (P =.050). These findings suggest a tendency for concurrent alterations of the cell cycle regulators p27, p53, and Rb or p16 in DLBCL, which might result in impaired tumor growth control. Indeed, the analysis of the combined p27/p53/Rb/p16 expression status with respect to the proliferation profile showed that (1) three alterations in the combined p27/p53/Rb/p16 status (i.e., low/null P27 expression, increased expression of p53, and decreased expression of Rb or p16) were significantly correlated with increased expression of cyclin B1 (P =.005) and (2) two or three alterations were significantly correlated with increased expression of cyclin A (P =.014). These findings suggest combined impairment of a complex cell-cycle control network involving the CDK inhibitor p27, the P53 pathway, and the Rb1 pathway, which exerts a cooperative effect resulting in enhanced tumor cell proliferation.     

Oligomerization of polyalanine expanded PABPN1 facilitates nuclear protein aggregation that is associated with cell death.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)(6) repeat to (GCG)(8-13) in the PABPN1 gene, which results in the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of the protein. Mutated PABPN1 (mPABPN1) is able to induce nuclear protein aggregation and form filamentous nuclear inclusions, which are the pathological hallmarks of OPMD. PABPN1, when bound to poly(A) RNA, forms both linear filaments and discrete-sized, compact oligomeric particles in vitro. In the absence of poly(A) RNA, PABPN1 can form oligomers. Here we report that: (i) oligomerization of PABPN1 is mediated by two potential oligomerization domains (ODs); (ii) inactivating oligomerization of mPABPN1 by deletions of 6-8 amino acids in either of the ODs prevents nuclear protein aggregation; (iii) expression of mPABPN1 in COS-7 cells is associated with cell death; and (iv) preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. These findings suggest that oligomerization of PABPN1 plays a crucial role in the formation of OPMD nuclear protein aggregation, while the expanded polyalanine stretch is necessary but not sufficient to induce OPMD protein aggregation, and that the nuclear protein aggregation might be toxic and cause cell death. These observations also imply that inactivation of oligomerization of mPABPN1 might be a useful therapeutic strategy for OPMD.     

杀菌性通透性增强蛋白对大肠杆菌脓毒症小鼠的保护作用

本文观察了ＢＰＩ对大肠杆菌脓毒症小鼠预后的影响及其可能机制。研究结果显示〉ＢＰＩ治疗组动物７２小时存活率明显高于生理盐水对照组；注菌后０．５，１ｈ，ＢＰＩ组血清内毒素水平明显低于生理盐水对照组；注菌后１．５ｈ，ＢＰＩ组血清ＴＮＦα峰值也明显低于ＮＳ对照组；但两组间血液细菌计数在注菌后０．５，１．５和３ｈ均明显差异。     

Y-position cysteine substitution in type I collagen (alpha1(I) R888C/p.R1066C) is associated with osteogenesis imperfecta/Ehlers-Danlos syndrome phenotype

The most common mutations in type I collagen causing types II-IV osteogenesis imperfecta (OI) result in substitution for glycine in a Gly-Xaa-Yaa triplet by another amino acid. We delineated a Y-position substitution in a small pedigree with a combined OI/Ehlers-Danlos Syndrome (EDS) phenotype, characterized by moderately decreased DEXA z-score (-1.3 to -2.6), long bone fractures, and large-joint hyperextensibility. Affected individuals have an alpha1(I)R888C (p.R1066C) substitution in one COL1A1 allele. Polyacrylamide gel electrophoresis (PAGE) of [(3)H]-proline labeled steady-state collagen reveals slight overmodification of the alpha1(I) monomer band, much less than expected for a substitution of a neighboring glycine residue, and a faint alpha1(I) dimer. Dimers form in about 10% of proband type I collagen. Dimer formation is inefficient compared to a possible 25%, probably because the SH-side chains have less proximity in this Y-position than when substituting for a glycine. Theoretical stability calculations, differential scanning calorimetry (DSC) thermograms, and thermal denaturation curves showed only weak local destabilization from the Y-position substitution in one or two chains of a collagen helix, but greater destabilization is seen in collagen containing dimers. Y-position collagen dimers cause kinking of the helix, resulting in a register shift that is propagated the full length of the helix and causes resistance to procollagen processing by N-proteinase. Collagen containing the Y-position substitution is incorporated into matrix deposited in culture, including immaturely and maturely cross-linked fractions. In vivo, proband dermal fibrils have decreased density and increased diameter compared to controls, with occasional aggregate formation. This report on Y-position substitutions in type I collagen extends the range of phenotypes caused by nonglycine substitutions and shows that, similar to X- and Y-position substitutions in types II and III collagen, the phenotypes resulting from nonglycine substitutions in type I collagen are distinct from those caused by glycine substitutions.     

Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides.

Cell surface proteins SspA and SspB in Streptococcus gordonii and SpaP in Streptococcus mutans are members of the antigen I/II family of polypeptides produced by oral streptococci. These proteins are adhesins and mediate species-specific binding of cells to a variety of host and bacterial receptors. Here we show that antigen I/II polypeptides are involved in the attachment of oral streptococci to collagen and that they also determine the ability of these bacteria to invade human root dentinal tubules. Wild-type S. gordonii DL1 (Challis) cells showed heavy invasion of tubules to a depth of approximately 200 microm, whereas the abilities of cells of isogenic mutant strains OB220 (sspA) and OB219 (sspA sspB) to invade were 50 and >90% reduced, respectively. Likewise, wild-type S. mutans NG8 cells invaded dentinal tubules, whereas cells of isogenic mutant strain 834 (spaP) did not. The invasive abilities of strains OB220 and OB219 were restored by heterologous expression of S. mutans SpaP polypeptide in these strains. The extents of tubule invasion by various wild-type and mutant strains correlated with their levels of adhesion to type I collagen, a major component of dentin. Furthermore, S. gordonii DL1 cells exhibited a growth response to collagen by forming long chains. This was not shown by ssp mutants but was restored by the expression of SpaP in these cells. The production of SspA polypeptide by S. gordonii DL1, but not production of SspB polypeptide by strain OB220 (sspA), was enhanced in the presence of collagen. These results are the first to demonstrate that antigen I/II family polypeptides bind collagen and mediate a morphological growth response of streptococci to collagen. These antigen I/II polypeptide activities are critical for intratubular growth of streptococci and thus for establishment of endodontic infections.     

Neovascularization in aged mice: delayed angiogenesis is coincident with decreased levels of transforming growth factor beta1 and type I collagen.

Angiogenesis, the growth of new vessels from existing microvasculature, is delayed in aged animals. In this study we asked whether this impairment might be due, in part, to changes in the expression of a growth factor, transforming growth factor-beta1 (TGF-beta1), and a matrix protein, type I collagen, which have been shown to regulate angiogenesis in vivo. We implanted polyvinyl alcohol sponges subcutaneously in the dorsa of young and aged mice and examined the sponges 7 to 21 days later for the presence of invasive fibrovascular bundles. Blood vessel ingrowth and proliferative activity were assessed by immunostain for von Willebrand factor and Ki-67, respectively. The fibrovascular bundles were also analyzed for TGF-beta1 and type I collagen. Relative to young mice, angiogenic invasion of sponges in aged mice was similar at 7 days, was diminished significantly (70%) at 14 days, but was again similar by 21 days after implantation. The expression of TGF-beta1 and type I collagen mRNA and protein in fibrovascular bundles was coincident but was also delayed (42 to 47%) at 14 days in the aged mice. Moreover, levels of active TGF-beta1 were decreased (48%) in the sera of aged relative to young mice. The delay in angiogenesis in aged mice was thus associated with decreased expression of TGF-beta1 and type I collagen by neovascular bundles. We conclude that changes in the levels of growth factors and proteins in the extracellular matrix contribute to impaired angiogenesis in aging.