Genome-wide association analysis for susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in US Holsteins.

Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP and concurrently modifying management to reduce the likelihood of infection. The current study was motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. Two separate GWAS analyses were conducted, the first using 897 genotyped Holstein artificial insemination sires with phenotypes derived from incidence of MAP infection among daughters based on milk ELISA testing records. The second GWAS analysis was a case-control design using US Holstein cows phenotyped for MAP infection by serum ELISA or fecal culture tests. Cases included cows positive for either serum ELISA, fecal culture, or both. Controls consisted of animals negative for all tests conducted. A total of 376 samples (70 cases and 306 controls) from a University of Minnesota Johne's management demonstration project and 184 samples (76 cases and 108 controls) from a Michigan State University study were used. Medium-density (sires) and high-density (cows) genotype data were imputed to full genome sequence for the analyses. Marker-trait associations were analyzed using the single-step (ss)GWAS procedure implemented in the BLUPF90 suite of programs. Evidence of significant genomic contributions for susceptibility to MAP infection were observed on multiple chromosomes. Results were combined across studies in a meta-analysis, and increased support for genomic regions on BTA7 and BTA21 were observed. Gene set enrichment analysis suggested pathways for antigen processing and presentation, antimicrobial peptides and natural killer cell-mediated cytotoxicity are relevant to variation in host susceptibility to MAP infection, among others. Genomic prediction was evaluated using a 5-fold cross-validation, and moderate correlations were observed between genomic breeding value predictions and daughter averages (∼0.43 to 0.53) for MAP infection in testing data sets. These results suggest that genomic selection against susceptibility to MAP infection is feasible in Holstein cattle.

The role of subchondral bone, and its histomorphology, on the dynamic viscoelasticity of cartilage, bone and osteochondral cores.

OBJECTIVE: Viscoelastic properties of articular cartilage have been characterised at physiological frequencies. However, studies investigating the interaction between cartilage and subchondral bone and the influence of underlying bone histomorphometry on the viscoelasticity of cartilage are lacking. METHOD: Dynamic Mechanical Analysis (DMA) has been used to quantify the dynamic viscoelasticity of bovine tibial plateau osteochondral cores, over a frequency sweep from 1 to 88 Hz. Specimens (approximately aged between 18 and 30 months) were neither osteoarthritic nor otherwise compromised. A maximum nominal stress of 1.7 MPa was induced. Viscoelastic properties of cores have been compared with that of its components (cartilage and bone) in terms of the elastic and viscous components of both structural stiffness and material modulus. Micro-computed tomography scans were used to quantify the histomorphological properties of the subchondral bone. RESULTS: Opposing frequency-dependent loss stiffness, and modulus, trends were witnessed for osteochondral tissues: for cartilage it increased logarithmically (P < 0.05); for bone it decreased logarithmically (P < 0.05). The storage stiffness of osteochondral cores was logarithmically frequency-dependent (P < 0.05), however, the loss stiffness was typically frequency-independent (P > 0.05). A linear relationship between the subchondral bone plate (SBP) thickness and cartilage thickness (P < 0.001) was identified. Cartilage loss modulus was linearly correlated to bone mineral density (BMD) (P < 0.05) and bone volume (P < 0.05). CONCLUSION: The relationship between the subchondral bone histomorphometry and cartilage viscoelasticity (namely loss modulus) and thickness, have implications for the initiation and progression of osteoarthritis (OA) through an altered ability of cartilage to dissipate energy.

Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy.

OBJECTIVE: This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy. METHOD: In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-ß (TGFß) following 21 days of culture in either growth or chondrogenic media. RESULTS: In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFß. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets. CONCLUSION: The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.

Debonding force and deformation of two multi-stranded lingual retainer wires bonded to incisor enamel: an in vitro study.

The aim of the study was to examine, in vitro, the effect of a vertical force on the debond force and deformation of two multi-stranded wires bonded to the lingual enamel of lower incisor teeth. Two different stainless steel wires were used, 0.016 × 0.022 inch (Bond-A-Braid® Reliance Orthodontic Products) and a three-stranded 0.0175 inch wire (Ortho Technology). An in vitro model was used to simulate a vertical force at the interdental wire. Twenty-six pairs of incisors were placed in two groups. A 15 mm length of wire was bonded to the lingual surfaces of each pair of incisors using a common bonding technique. A vertical force was applied to the midpoint of the interdental wire, using an Instron universal testing machine. The failure characteristics examined included the maximum force for debond, the degree of wire deformation, and the site of failure. Significance was predetermined at P < 0.05 and multiple comparisons indicated no significant differences (P = 0.147) in the initial mean bond strength between the 0.0175 inch (41.44 N) and 0.016 × 0.022 inch (37.70 N) wires. The main failure type for both the initial and second debond events was fracture of composite bond at the wire-composite interface, cohesive failure. Both wires exhibited similar mean degrees of deflection of 1.30 and 1.51 mm for the 0.0175 inch and 0.016 × 0.022 inch wires, respectively. Rebonding to enamel resulted in significantly lower (P = 0.001) mean bond strength for both wires, 0.0175 inch (13.86 N) and 0.016 × 0.022 inch (14.17 N) in comparison with the initial bond strength. Rebonding to previously bonded enamel may be unpredictable and may lead to higher failure rates of bonded lingual retainers.

Maxillary premolar resorption by canines: three case reports.

Three unusual cases of maxillary premolar root resorption are reported. Three teenage patients were referred to the orthodontic department for management of ectopic maxillary canines. Radiographic examination revealed unilateral premolar root resorption in all three patients. This represents an unusual finding. Whereas the prevalence of maxillary lateral incisor root resorption secondary to palatally ectopic canines has been reported, the prevalence of premolar root resorption is unknown. This report discusses the findings in the context of the available literature. The postulated aetiology and the need for early diagnosis are highlighted.

Stabilisation of the unstable fractured zygomatic arch with a Kirschner wire.

Although relatively uncommon, isolated fractures of the zygomatic arch can sometimes be difficult to stabilise following reduction. We present a simple method of stabilisation using a Kirschner wire.

Schinzel-Giedion syndrome: interesting facial and orodental features, and dental management.

Schinzel-Giedion syndrome comprises multiple congenital anomalies. The orofacial features include coarse facies, frontal bossing, ocular hypertelorism, anterior open bite and macrodontia. Two cases are presented in which the presence of specific craniofacial anomalies with bilateral hydronephrosis confirmed the diagnosis. In one patient, bottle-feeding was associated with caries in maxillary central and lateral incisors, but the second patient was permanently tube fed and did not experience any dental caries. Clinical management of these patients requires a coordinated approach from a team of medical and dental specialists.

Modulation of cell interactions with extracellular matrix by lysophosphatidic acid and sphingosine 1-phosphate.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (SPP) are lipid mediators released upon platelet activation. The concentration of LPA in serum is estimated at 1-10 microM whereas the concentration in plasma is considerably less. The SPP concentration in serum is 0.5 microM, approximately two-fold higher than the plasma concentration. The lipids are present during tissue injury and promote cellular processes involved in wound repair. LPA and SPP have multiple effects on cells, many of which are pertinent to wound healing and require that the cells interact in some fashion with components of the extracellular matrix. This review focuses on modulation of cell adhesion, cell migration, collagen gel contraction, and fibronectin matrix assembly by LPA and SPP.

Contraction of collagen matrices mediated by alpha2beta1A and alpha(v)beta3 integrins.

The (beta)1-null fibroblastic cell line GD25 and its derivatives were studied to gain an understanding of the roles of (beta)1 and (beta)3 integrins in the initial (1-hour) contraction of collagen gels. Stable transfectants of GD25 cells expressing the (beta)1A splice variant of (beta)1 ((beta)1A-GD25) did not express (alpha)2(beta)1A and did not adhere to collagen. After transfection of (alpha)2 into (beta)1A-GD25 cells, the (alpha)2(beta)1A-GD25 transfectants contracted collagen gels in the presence of serum, whereas (beta)1A-GD25 cells did not. The GD25 parental cells, however, also contracted collagen gels. Collagen gel contraction by GD25 cells was blocked by antibodies to (alpha)v(beta)3 or a RGD-containing peptide, indicating that (alpha)v(beta)3 is the integrin responsible for mediation of contraction by GD25 cells. Collagen gel contraction by (alpha)2(beta)1A-GD25 cells was not inhibited by antibodies to (alpha)v(beta)3 or RGD-containing peptide, but was inhibited by anti-(alpha)2 antibody. Flow cytometry demonstrated negligible expression of (alpha)v(beta)3 by (beta)1A-GD25 and (alpha)2(beta)1A-GD25 cells when compared to GD25 cells. Platelet derived growth factor (PDGF) and sphingosine-1-phosphate (S1P) enabled gel contraction by (alpha)2(beta)1A-GD25 and GD25 cells, respectively, in the absence of serum. PDGF-stimulated contraction by (alpha)2(beta)1A-GD25 cells was attenuated in the presence of inhibitors of phosphatidylinositol-3-kinase whereas such inhibitors had no effect on S1P-stimulated contraction by GD25 cells. These experiments using the (beta)1-null GD25 cells and (beta)1A and (alpha)2(beta)1A transfectants demonstrate that (alpha)2(beta)1A and (alpha)v(beta)3 independently mediate collagen gel contraction and are regulated by different serum factors and signaling pathways.

Cervical interbody fusion cages. An animal model with and without bone morphogenetic protein.

STUDY DESIGN: The Alpine goat model for multilevel anterior cervical discectomy and fusion was used to analyze the use of an intervertebral fusion device to promote an arthrodesis after anterior cervical discectomy. Comparisons were drawn with biomechanical, histologic, and radiographic data. OBJECTIVES: To analyze the use of an intervertebral fusion device, with and without a bone graft substitute, to promote an arthrodesis anterior cervical discectomy. SUMMARY OF BACKGROUND DATA: In previous studies, the goat cervical spine has proven to be an excellent model for examining the healing of fusions using bone grafts, instrumentation, or bone substitutes. METHODS: Three-level anterior cervical dissectomies were performed on 21 mature Alpine goats. Three treatment groups of seven goats each were used. Group I used a standard titanium cervical BAK device filled with autogenous bone graft. Group II used a hydroxyapatite-coated BAK device filled with autogenous bone graft. Group III used a BAK device filled with recombinant human bone morphogenetic protein-2. RESULTS: Radiographically, no cages became displaced. Lucencies were seen around 3 of the 21 cages in Group 1, 4 cages in Group II, and none in Group III. Fluorochrome analysis revealed that the recombinant human bone morphogenetic protein-2-filled cages had an accelerated rate of bone growth around and through each cage-vertebral body interface at 3 weeks. A successful arthrodesis was also more likely with a recombinant human bone morphogenetic protein-2-filled cage (95%) than the hydroxyapatite-coated (62%) or the standard (48%) cage. Biomechanical stiffness testing did not reveal any statistically significant differences between the three groups. There was a tendency for successfully arthrodesed interspaces to be stiffer than those that were not. CONCLUSIONS: The use of a threaded intervertebral fusion cage, with or without hydroxyapatite coating, filled with autogenous bone graft provides a fusion rate that is slightly better than those previously reported using autogenous interbody bone grafts with or without plate stabilization. Recombinant human bone morphogenetic protein-2-filled cages resulted in a much higher arthrodesis rate and accelerated bone formation compared with either autogenous bone-filled BAK devices, or autogenous interbody bone grafts with or without plate stabilization.

Effect of antisense oligonucleotides on cytokine release from human keratinocytes in an in vitro model of skin.

ISIS 1082, a phosphorothioate oligonucleotide 21 nucleotides in length targeted to the translation initiation codon of herpes simplex virus (HSV) type 1 and 2 virion capsid protein, has been shown to inhibit HSV-1 replication in vitro. The effects of ISIS 1082, its phosphodiester congener, ISIS 1049, and analogs consisting of 2' methoxy and 2' propoxy phosphodiesters and phosphorothioates on IL-1 alpha release and viability were evaluated in a three-dimensional in vitro skin model consisting of neonatal keratinocytes and fibroblasts. This in vitro system displays many of the functional and metabolic properties of a differentiated epidermis and can be induced to specifically release IL-1 alpha in response to a mixture of lipopolysaccharide and phorbol myristate acetate. Incubation of the skin model with 250 to 1000 microM concentrations of ISIS 1082 and its 2' methoxy and propoxy phosphorothioate analogs resulted in a concentration-dependent increase of cytokine release with minimal effects on cellular viability, as measured by the Neutral Red assay. This response was confirmed in primary keratinocytes, which were also shown to secrete IL-1 alpha into media supernatants after incubation with phosphorothioate oligomers. These data suggest that the IL-1 alpha released from keratinocytes in response to ISIS 1082 may contribute to the inflammatory and immune cell response seen in vivo.

In vitro pharmacokinetics of phosphorothioate antisense oligonucleotides.

ISIS 2105 (Afovirsen), a 20-mer phosphorothioate oligonucleotide that inhibits the production of a gene product essential to the growth of human papillomavirus, is in phase II clinical trials for the treatment of genital warts induced by human papillomavirus-6 and human papillomavirus-11. The uptake, subcellular distribution and metabolism of ISIS 2105 and three other similar length phosphorothioates have been studied in a variety of cell lines. Our experiments indicated that ISIS 2105 and other phosphorothioates are internalized and distributed in a time-, temperature-, concentration-, sequence- and cell line-dependent manner. Cell association was also influenced by the tissue culture medium. Several different analytical techniques revealed that phosphorothioates were more rapidly degraded in vitro than previously reported. These data suggest that phosphorothioate oligonucleotide uptake and stability observed in tissue culture can vary as a function of cellular assay conditions and analytical methods used. Comparison of these results with those obtained in vivo suggests that the pharmacokinetic behavior of this class of compounds cannot necessarily be predicted from in vitro studies.

Anterior cervical discectomy and fusion using a porous hydroxyapatite bone graft substitute.

OBJECTIVES: This study analyzed the use of a coral hydroxyapatite bone substitute for use in ACDF both with and without an anterior cervical plate. STUDY DESIGN: The healing of multilevel anterior cervical fusions was tested using a goat model. Comparisons were drawn with histologic, radiographic, and biomechanical test data. METHODS: Forty-nine mature alpine goats had three-level anterior discectomies performed. Seven treatment groups of seven goats each were used; Group I with no fusion, Group IIa having tricortical iliac crest autograft, Group IIb having autograft plus an anterior plate, Group IIIa having tricortical iliac crest fresh-frozen allograft, Group IIIb having allograft plus an anterior plate, Group IVa having rectangular-shaped implants of porous hydroxyapatite, and Group IVb having ProOsteon 500 implants with an anterior cervical plate. RESULTS: Histologically, at 12 weeks 48% of the ProOsteon (Interpore, Irvine, CA) implants were rated as incorporated, 10% as possessing a fibrous gap, 29% as collapsed, and 14% as extruded. Anterior cervical plating improved the results with 71% of the implants showing good incorporation, 24% with collapse, and 5% with a fibrous gap. These histologic results compare favorably with autogenous bone and are improved over allograft bone. Fluorochrome analysis showed that none of the implants had complete turnover with host bone, but that all possessed peripheral creeping substitution with cutting cones of new bone formation at 12 weeks. Biomechanically, the spines using the ProOsteon implant were less stiff in torsion than autograft, but equal in stiffness to allograft. Flexion-extension neutral zone stiffness was lower in the ProOsteon implant group than either allograft or autograft. CONCLUSIONS: The use of a coral-based hydroxyapatite bone graft substitute for anterior cervical fusions led to significant rates of implant collapse at 12 weeks but showed excellent biologic compatibility with good early creeping substitution of the implant by host bone. The concomitant use of an anterior cervical plate with the implant prevents extrusion.

Anterior cervical discectomy, fusion, and plating. A comparative animal study.

Thirty-five goats were used as animal models for three-level anterior cervical discectomy and fusion. The goats were divided equally into five experimental groups: Group I, three-level anterior cervical discectomy without fusion; Group IIa, three-level discectomy with autogenous bone (Smith-Robinson technique); Group IIb, autogenous bone grafting plus anterior plate application; Group III, three-level discectomy and fusions with fresh frozen allograft bone; Group IIIa, graft only; and Group IIIb, anterior plating. The goats were killed, and spines were removed and analyzed for evidence of fusion. Radiographic union was judged to have occurred in 0% of Group I disc spaces, 48% of Group IIa, 52% of Group IIb, and 38% of Groups IIIa and IIIb. Histologic fusion was judged to have occurred in 0% of Group I specimens, 38% of Group IIa, 45% of Group IIb, 0% of Group IIIa, and 19% of Group IIIb. The histologic fusion rate was significantly higher in Groups IIa and IIb than in Groups I and IIIa. There was no statistically significant increase in the histologic fusion rate between goats with anterior cervical plating and goats without plating. Biomechanically, the spines in Groups IIb and IIIb, ie, those with anterior plates applied, were stiffer in axial load, torsion, and flexion/extension. Peri-end-plate vascularity was significantly diminished in those groups that had anterior plates applied. Qualitative analysis of fluorochrome labels showed that autografts revascularized more rapidly than did allografts in both the nonplated and plated groups. Histomorphometric analysis failed to reveal any significant device-related osteopenia in those vertebrae spanned by the anterior plate. We found that although autograft bone led to a significantly higher rate of union than did allograft bone, the addition of anterior plate fixation did not significantly increase union rate. Biomechanical rigidity was significantly increased in all modes of testing by the use of an anterior plate. The decreased vascular response seen in spines that underwent plating may be responsible for the lack of increased union. The increased rigidity found with anterior plating supports its use in traumatic conditions. The failure, however, to increase significantly the union rate in this model fails to lend support to the use of anterior cervical plating for degenerative discectomy and fusion procedures.

Pedicle screw pullout strength. Correlation with insertional torque.

This study was designed to correlate several parameters regarding pedicle screw bone/metal interface strength. The insertional torque measured during tapping and placement of pedicle screws was correlated with the bone mineral density of the vertebral body, the dimensions of the pedicle, the method of preparation of the pedicle, and the amount of load and number of cycles to failure of the bone/metal interface. Thirty human cadaveric lumbar vertebrae were instrumented with 6.5-mm pedicle screws. The maximum torque achieved during insertion was digitally recorded. A cyclic pedicle screw pullout test was performed. A linear correlation existed between both the insertional torque when tapping or when inserting a screw and the number of cycles to ultimate pedicle screw pullout. An inverse linear relationship was found between the pedicle width and cycles to failure. There was no linear correlation found when comparing the number of cycles to failure to bone mineral density. These findings suggest that insertional torque is a good predictor of bone-metal interface failure. Bone mineral density of the vertebral body was less effective as a predictor of failure. Smaller pedicle width correlated with increased insertional torque and cycles to failure. This may explain why patients with osteoporosis on radiography may still obtain stable fixation with pedicle screws. Other factors, such as pedicle dimensions and shape, affect screw purchase as much as vertebral body bone density. Insertional torque less than 4.0 inch-pounds led to early pedicle screw pullout. This study forms the basis for the authors' clinical use of an instrumented torque screwdriver to measure insertional torque in the operating room.

Spinal instrumentation for metastatic disease: in vitro biomechanical analysis.

A calf spine corpectomy model was employed to compare the ability of various spinal instrumentation techniques to restore stability after destruction of the anterior and middle columns. The constructs tested were an intact specimen, a Harms titanium cage, a cross-linked rectangular anterior Texas Scottish Rite Hospital construct, posterior segmental Luque rods combined with a cross-linked rectangular anterior TSRH construct, posterior Luque rods alone, cross-linked posterior Luque rods, and posterior Luque rods embedded in polymethyl-methacrylate (PMMA). When compared to the intact state, only the combined anterior/posterior construct and the anterior TSRH construct were able to restore axial, sagittal, and torsional stiffnesses to levels equal to or higher than control levels. Embedding Luque rods in PMMA provided greater stiffness than either Luque rods alone or cross-linked Luque rods. If the anterior and middle columns of the spine are incompetent, as frequently occurs with metastatic disease, the goal of surgical treatment is to restore spinal stability while decompressing the neural elements. Either anterior or posterior techniques may be used. The findings of this study imply that commonly used spinal instrumentation constructs differ in their ability to restore axial, sagittal, and torsional stiffnesses to normal levels.

Anterior cervical discectomy and fusion. A comparison of techniques in an animal model.

An animal model for three-level anterior cervical discectomy and fusion was established in the goat. Twenty-one goats underwent surgery, with seven goats in each of three experimental groups. In Group I, all seven goats underwent three-level anterior cervical discectomy without fusion. In Group II, each of the seven goats had a three-level discectomy with autogenous bone performed according to the Smith-Robinson technique. In Group III, fresh-frozen allograft bone was used for each of the three-level discectomy and fusion. Each goat was then killed after 12 weeks. Analysis consisted of radiographic review, fluorochrome labeling, biomechanical rigidity and flexion and extension, axial compressive load, and torsion. Histologic analysis was also performed for evidence of fusion and vertebral body histomorphometric analysis. The analysis of results showed that radiographic union was judged to have occurred in 0 of 21 Group I disc spaces, 10 of 21 Group II disc spaces, and 8 of 21 Group III disc spaces. Histologic fusion was judged to have occurred in 0 of 21 Group I goats, 10 of 21 Group II goats, and 0 of 21 Group III goats. The histologic fusion rate was significantly higher in Group II than either Group I or Group III. Biomechanically, the spines that had autogenous bone grafting (Group II) were significantly stiffer in compressive axial load and in extension. Both Group II and Group III were stiffer in flexion than Group I. An evaluation of the peri-endplate vascularity showed that the vascularity measured 10.4% in Group I, 16.7% in Group II, and 8.5% in Group III.(ABSTRACT TRUNCATED AT 250 WORDS)

Citrate is a major component of snake venoms.

Citrate has been identified as a major component of snake venoms by gas liquid chromatography and mass spectrometry. The venoms of Bothrops asper, Crotalus atrox, Crotalus viridis viridis, Crotalus adamanteus, Sistrurus miliarius barbouri, Crotalus horridus horridus, Agkistrodon contortrix mokasen, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus contain citrate at concentration levels which can serve as effective buffers. Calcium, magnesium, zinc, iron, sodium and potassium salts of citrate would be constituents of venom.