Total hip arthroplasty in patients 16 years of age or younger.

PURPOSE: Total hip arthroplasty (THA) is rarely recommended in skeletally immature patients. The goal of the present study was to report our short- to mid-term results of THA in a series of children aged 16 years or younger, including clinical outcomes and post-operative complications, signs of radiographic loosening of the prostheses, and revision rate. METHODS: The 18 children (two male and 16 female patients, 24 hips) underwent cementless THA at a mean age of 14.6 years (11 to 16). Five patients had a bilateral, one-stage surgical procedure. Clinical assessment of these hips used the Merle d'Aubigné et Postel scale modified by Charnley to facilitate assessment of the function of walking. Clinical and radiographic follow-up was conducted at six weeks, six months and then yearly for the first three years. All post-operative complications were recorded. RESULTS: No intra-operative or early post-operative complications occurred. At a mean follow-up of 3.8 years (1 to 8), all patients had greatly improved pain and function scores. All children in the present study improved from severely impaired gait, including four children who were wheelchair-bound, to completely unrestricted gait. All hips demonstrated good alignment with no evidence of wear or radiographic lucencies. No revision of components has been required. One patient had persistent adductor contracture which was addressed with adductor tendon release. CONCLUSIONS: THA is a successful procedure for unsalvageable hip arthritis in children at a mean follow-up of 3.8 years. Long-term follow-up will be needed to determine implant longevity of the components in these children.

Proximal versus distal screw placement for biceps tenodesis: a biomechanical study.

PURPOSE: To assess the maximum and end torque of a fourth-generation composite humerus model with no screw inserted or with a screw inserted in the distal (subpectoral) position or proximal (suprapectoral) position. METHODS: 24 large-size, fourth-generation composite humeri were randomised to the control (n=8), proximal (n=8), or distal (n=8) group. For the latter 2 groups, an 8-mm-head interference screw (7x25 mm) was inserted at 1 cm proximal and 1 cm distal to the superior aspect of the insertion of the pectoralis major tendon, respectively. The maximum and end torque of each humerus was assessed. RESULTS: Respectively for the control, proximal, and distal groups, the maximum torque was 81.8, 78.7, and 74.3 Nm, and the end torque was 80.7, 78.6, and 71.8 Nm; only the difference between control and distal groups was significant (p=0.005 for maximum torque and p=0.033 for end torque). All fractures in both control and proximal groups involved the distal 1/3 humerus. In the distal group, the fractures involved either the distal 1/3 humerus (n=6) or the screw-hole (n=2); the difference between the 2 types of fracture was not significant in terms of maximum torque (75.7 vs. 70.0, p=0.086) or end torque (75.3 vs. 61.4, p=0.40). CONCLUSION: Compared with proximal placement of an interference screw, distal placement decreased the maximum torque (though not significantly) and may increase the risk of proximal humeral fracture.

Femoroacetabular impingement osteoplasty: is any resected amount safe? A laboratory based experiment with sawbones.

There is an increased risk of fracture following osteoplasty of the femoral neck for cam-type femoroacetabular impingement (FAI). Resection of up to 30% of the anterolateral head-neck junction has previously been considered to be safe, however, iatrogenic fractures have been reported with resections within these limits. We re-evaluated the amount of safe resection at the anterolateral femoral head-neck junction using a biomechanically consistent model. In total, 28 composite bones were studied in four groups: control, 10% resection, 20% resection and 30% resection. An axial load was applied to the adducted and flexed femur. Peak load, deflection at time of fracture and energy to fracture were assessed using comparison groups. There was a marked difference in the mean peak load to fracture between the control group and the 10% resection group (p < 0.001). The control group also tolerated significantly more deflection before failure (p < 0.04). The mean peak load (p = 0.172), deflection (p = 0.547), and energy to fracture (p = 0.306) did not differ significantly between the 10%, 20%, and 30% resection groups. Any resection of the anterolateral quadrant of the femoral head-neck junction for FAI significantly reduces the load-bearing capacity of the proximal femur. After initial resection of cortical bone, there is no further relevant loss of stability regardless of the amount of trabecular bone resected. Based on our findings we recommend any patients who undergo anterolateral femoral head-neck junction osteoplasty should be advised to modify their post-operative routine until cortical remodelling occurs to minimise the subsequent fracture risk.

Evaluation of focal choroidal excavation in the macula using swept-source optical coherence tomography.

PURPOSE: To evaluate imaging findings of patients with focal choroidal excavation (FCE) in the macula using swept-source optical coherence tomography (SS-OCT) and correlate it clinically. METHODS: Prospective observational case series. Eleven consecutive patients (12 eyes) with FCE were described. Data on demographics and clinical presentation were collected and imaging findings (including color photography, fundus autofluorescence imaging, fluorescein angiography, indocyanine green angiography, spectral-domain optical coherence tomography, and SS-OCT) were analyzed. RESULTS: The primary diagnosis was epiretinal membrane (two eyes), choroidal neovascularization (one eye), polypoidal choroidal vasculopathy (three eyes), central serous chorioretinopathy (one eye), and dry age-related macular degeneration (two eyes). Eleven out of 12 of the lesions were conforming. One presented with a non-conforming lesion that progressed to a conforming lesion. One eye had multiFCE and two had two overlapping choroidal excavations. Using the SS-OCT, we found the choroid to be thinned out at the area of FCE but sclera remained normal. The choroidal tissue beneath the FCE was abnormal, with high internal reflectivity and poor visualization of choroidal vessels. There was loss of contour of the outer choroidal boundary that appeared to be pulled inward by this abnormal choroidal tissue. A suprachoroidal space was noted beneath this choroidal tissue and the choroidal-scleral interface was smooth. Repeat SS-OCT 6 months after presentation showed the area of excavation to be stable in size. CONCLUSION: FCE can be associated with epiretinal membrane, central serous chorioretinopathy, and age-related macular degeneration. The choroid was thinned out in the area of FCE.

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates.

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo.

BACKGROUND: The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis. OBJECTIVES: We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. METHODS: (i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively. RESULTS: (i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation. CONCLUSIONS: We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.

An object transport system using flexural ultrasonic progressive waves generated by two-mode excitation.

An object transport system using low amplitude and high frequency progressive waves generated by two-mode excitation is presented. A theoretical model for the system was developed using normal mode expansion and the modal participation factor. To identify the factors that affect the transport speed, the changes with the mass of objects on the beam, the input power, the phase difference, and the excitation frequency were experimentally investigated. With a power input of 40 W, a transport speed of 10 cm/s was obtained for an object weighing 30 g. The tests indicate that, not only the phase difference but also the excitation frequency, were the dominant factors in determining the transport speed and direction. Specifically, when the excitation frequency was chosen to be at the exact midpoint of the two modes, the object stopped moving. A slight change of frequency in either direction resulted in change of object transport direction. For actual factory application, a simple stop-go and tracking control using the General Purpose Interface Bus (GPIB) were implemented.

Active constituents against HIV-1 protease from Garcinia mangostana.

The ethanol extract of Garcinia mangostana L. (Guttiferae) showed potent inhibitory activity against HIV-1 protease. The activity-guided purification of the extract resulted in the isolation of two active, known compounds. The chemical structures of the isolated compounds were established by spectroscopic analyses as mangostin (IC50 = 5.12 +/- 0.41 microM) and gamma-mangostin (IC50 = 4.81 +/- 0.32 microM). The type of inhibition by both compounds is noncompetitive.

Autoprocessing: an essential step for the activation of HIV-1 protease.

Human immunodeficiency virus type 1 (HIV-1) expresses its structural and functional proteins within Gag-Pol precursor polyproteins. Specific proteolytic processing of the precursors by the viral protease is critical for the maturation and infectivity of viral particles. To observe the influence of autoprocessing on the activation of recombinant HIV-1 protease, we constructed different HIV-1 protease forms, with or without the Phe-Pro bond directly upstream of the protease domain, and expressed them in Escherichia coli systems. We found that the presence of a short upstream sequence of the protease domain, which could generate the original N-terminus of the protease by autoproteolysis of the Phe-Pro bond, resulted in processing of active protease, whereas for a wild-type protease extended only with the initiator methionine, the proteolytic activity was not recovered. Our results suggested that autoprocessing of the direct upstream sequence of the protease domain is an essential step for the activation of recombinant HIV-1 protease in the E. coli expression system. Expression of HIV-1 protease as fusion proteins revealed that the existence of a fusion portion increased the accumulation of expressed protease by affecting its homotypic dimer formation.

Comparison of HIV-1 protease expression in different fusion forms.

Earlier observations showed that the expression of recombinant protease of human immunodeficiency virus type-1 (HIV-1 PR) was usually in a low level, and its proteolytic activity and hydrophobicity were believed to be toxic for the host cells. Various constructs were investigated that contained an N-terminal extended HIV-1 PR gene (PR107) in order to find a system which can express this protease in high level. The constructs of PR107 gene expressed as fusion proteins either with glutathione S-transferase (GST) by pGEX-PR107 or with maltose-binding protein (MBP) by pMAL-PR107 showed that the full length of fusion protein exhibited self-cleavage in E. coli. The results from expression experiments indicated that the size of the fusion portion does not affect the self-processing of fused HIV-1 PR to release its mature form, despite the attachment of only one subunit of the dimeric protease to GST or MBP. The construct, pET-PR107, under the control of strong bacteriophage T7 promoter system, did not show clear advantages for expression of this HIV-1 PR. Comparing these three constructs, the pGEX-PR107 system showed the highest expression level. Quantitative immuno-blotting indicated that the amount of HIV-1 PR expressed by pGEX-PR107 was twice that expressed by pMAL-PR107, and thrice that expressed by pET-PR107. More than 1 mg of pure HIV-1 PR from per liter culture of E. coli. DH5 alpha containing pGEX-PR107 can be obtained via the purification procedures [Biochem. Mol. Biol. International, (1995) 35:899-912].(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and purification of active form of HIV-1 protease from E.coli.

We have subcloned an N-terminal extended protease gene of human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame into expression vector pGEX-KG. A relatively high level of expression of recombinant HIV-1 protease (PR) was achieved with isopropyl beta-D-thiogalactoside (IPTG) induction and glucose supplement. An isolation method consisting of denaturation of protein and followed by refolding was developed for releasing this recombinant HIV-1 PR into the soluble phase since most of the expressed protease was initially present in insoluble inclusion bodies. High purity of this recombinant HIV-1 PR was obtained by sequential purification using Sephadex G-50 gel filtration and CM-23 cellulose cation exchange chromatography, yielding the protease more than 1 mg per liter culture. N-terminal amino acid sequence analysis showed that the recombinant HIV-1 PR underwent autocleavage from the fusion protein during expression. SDS-PAGE indicated that the molecular weight of this recombinant HIV-1 PR is 11 kDa. This recombinant HIV-1 PR showed proteolytic activity for the synthetic peptide substrates corresponding to the sequence at the Gag MA/CA and Pol p6*/PR junctions. The purified enzyme whose specific activity for the heptapeptide SQNYPIV was 848.7 nmol*min-1*mg protease-1 also processed recombinant polyprotein Gag41 as its substrate.

Mucoid Pseudomonas aeruginosa resists nonopsonic phagocytosis by human neutrophils and macrophages.

Mucoid Pseudomonas aeruginosa is an important respiratory pathogen in patients with cystic fibrosis, and once acquired is virtually impossible to eradicate. Although mucoid P. aeruginosa is generally believed to be resistant to phagocytosis, the mechanism is not understood fully. We studied the nonopsonic phagocytosis by human neutrophils or macrophages of eight mucoid/nonmucoid P. aeruginosa pairs (three isogenic and five "wild-type"). Mucoid strains were relatively resistant to nonopsonic phagocytosis but the nonmucoid types were phagocytosis-susceptible as assessed by visual inspection and chemiluminescence assays. The mucoid and nonmucoid variants had equal numbers of pili but different surface characteristics as determined by biphasic partitioning in polyethylene glycol and dextran. The mucoid exopolysaccharide of mucoid strains appears to alter the surface characteristics of P. aeruginosa thereby rendering them resistant to nonopsonic phagocytosis. The resistance of mucoid variants of P. aeruginosa to nonopsonic phagocytosis may provide a survival advantage to these bacteria early in the course of pulmonary infection before opsonic antibody and complement are present in respiratory secretions.

Nonopsonic phagocytosis of nonmucoid Pseudomonas aeruginosa by human neutrophils and monocyte-derived macrophages is correlated with bacterial piliation and hydrophobicity.

We have shown previously that some strains of Pseudomonas aeruginosa from patients with cystic fibrosis are phagocytized by human polymorphonuclear leukocytes in the absence of serum opsonins. The purpose of this study was to identify the bacterial features which render certain strains susceptible to nonopsonic phagocytosis. Three strains were phagocytized by human neutrophils and monocyte-derived macrophages, and two were not, as determined by luminol-enhanced chemiluminescence, visual inspection of stained smears, and bactericidal assay. Strains that were phagocytized formed pellicles when grown in static broth, but the phagocytosis-resistant strains did not. The phagocytosis-susceptible strains were more heavily piliated and more hydrophobic than the resistant strains. Bacteria exposed to heat (60 degrees C) or UV irradiation were depiliated, as assessed by electron microscopy, and rendered resistant to phagocytosis. When P. aeruginosa was grown on agar, it was piliated, hydrophobic, and susceptible to nonopsonic phagocytosis, but when grown to stationary phase in shaken broth, it was nonpiliated, less hydrophobic, and resistant to phagocytosis. It appears that nonopsonic phagocytosis of certain P. aeruginosa strains by human polymorphonuclear leukocytes and macrophages is facilitated by hydrophobic interactions which may be determined in part by pili.

Fimbriae (pili): molecular basis of Pseudomonas aeruginosa adherence.

Pseudomonas aeruginosa produces polar pili which promote the adherence of the organism to host mucosal surfaces and to blood-borne phagocytic cells such as polymorphonuclear leukocytes. Pseudomonas polar pili are flexible filaments of 52 A diameter and 2500 nm average length. They consist of a single type of protein subunit, pilin, of molecular weight 15,000, which is arranged in a helical mode of 5 subunits per turn and a pitch of 41 A. Purified whole pili, and anti-pilus antiserum both inhibited the interaction of Pseudomonas aeruginosa strains with human buccal epithelial cells and PMNs, suggesting that Pseudomonas adherence to these mammalian cells is pilus mediated. No correlation was found between the level of cell surface fibronectin on human buccal endothelial cells and the adherence of Pseudomonas bacteria. Pseudomonas adherence to buccal endothelial cells obtained from patients with cystic fibrosis was somewhat less than that to buccal endothelial cells obtained from healthy individuals. Fibronectin levels on buccal endothelial cells from patients with cystic fibrosis were not significantly different than those found on buccal endothelial cells from healthy individuals. Studies with peptide fragments derived from purified pili showed that only one peptide encompassing 23 amino acid residues at the C-terminus of the pilus protein was able to inhibit in vitro adherence of P. aeruginosa PAK to human buccal cells. This peptide domain was tentatively assigned the receptor-binding function.

Opsonin-independent phagocytosis of Pseudomonas aeruginosa.

Three nonmucoid revertant P. aeruginosa strains from cystic fibrosis patients were phagocytized by human polymorphonuclear leukocytes in the absence of serum. Phagocytosis was inhibited by D-mannose and by mannose-containing sugars. Bacteria killed by heat or ultraviolet irradiation or grown in shaken broth were devoid of pili and resistant to nonopsonic phagocytosis. The mucoid parents of two phagocytosis-susceptible nonmucoid revertant strains were resistant to nonopsonic phagocytosis. The nonmucoid revertants were more hydrophobic in nature than the mucoid parents, but they were comparably piliated. Nonopsonic phagocytosis of P. aeruginosa by human neutrophils appears to be mediated in part by pili. Other factors such as the mucoid coating of certain strains may interfere with this process.

Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa.

The mode of interaction of the polycationic aminoglycoside antibiotics with the surface of Pseudomonas aeruginosa cells was studied with the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The addition of the aminoglycoside gentamicin to intact cells in the presence of NPN led to a shift in the fluorescence emission maximum from 460 to 420 nm. At the same time the NPN fluorescence intensity increased fourfold. Gentamicin caused no such effects when added to outer membrane vesicles, suggesting that the increased fluorescence resulted from the interaction of gentamicin with intact cells. Gentamicin-promoted NPN uptake was inhibited by the divalent cations Mg2+ and Ca2+, but occurred in the absence of gentamicin transport across the inner membrane. Low concentrations of gentamicin (2 micrograms/ml) caused NPN fluorescence to increase over a period of 4 min in a sigmoidal fashion. At higher concentrations (50 micrograms/ml) the increase occurred within a few seconds. The final fluorescence intensity was almost independent of the gentamicin concentration. A centrifugation technique was used to demonstrate that gentamicin caused actual uptake of NPN from the supernatant. The initial rate of NPN uptake varied according to the gentamicin concentration in a sigmoidal fashion. Similar data were obtained for seven other aminoglycoside antibiotics. The data, when reanalyzed as a Hill plot, gave a series of lines with a mean slope (the Hill number) of 2.26 +/- 0.26, suggesting that the interaction of aminoglycosides with the cell surface to permeabilize it to NPN involved at least three sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudoassociation constant K, from the Hill plots and the minimal inhibitory concentrations for the eight antibiotics. These results are consistent with the concept that aminoglycosides interact as a divalent cation binding site on the P. aeruginosa outer membrane and permeabilize it to the hydrophobic prove NPN.