Signature Tagged Mutagenesis of Haemophilus influenzae identifies genes required for in vivo survival.

The pathogenic bacterium Haemophilus influenzae causes meningitis, epiglottitis, pneumonia, otitis media and other infections. To further understand the genetic basis of invasive disease and to inform about the bacterium's requirements in an in vivo environment, we analysed a library of 1632 insertional Tn1545 -Delta3 transposon mutants for their capacity to cause systemic infection in an animal model. We identified 25 genes that are potentially essential for H. influenzae invasive disease, and are candidates for further exploratory research. Seven of the genes encode hypothetical proteins, the function of six of which could be tentatively assigned on the basis of functional motifs and low homology to other bacterial genes. Eleven genes encode central metabolic enzymes or transporters; eight encode proteins that interact with DNA or modify other proteins; and four encode enzymes involved in the elaboration of classical virulence determinants. Two genes have no known function. Independent mutagenesis of six of the 25 genes and determination of the competitive index confirmed that these genes are important or essential to the organism in an in vivo environment. This genome-wide analysis has identified metabolic and other genes required during invasive disease, and the findings may lead to new interventions to prevent and treat H. influenzae infections.

Functional opsonic activity of human serum antibodies to inner core lipopolysaccharide (galE) of serogroup B meningococci measured by flow cytometry.

A recently described flow cytometric opsonophagocytic assay (OPA) was adapted to quantify the functional activity of serum antibodies specifically directed against serogroup B inner core lipopolysaccharide (LPS) of Neisseria meningitidis. The percentage of human peripheral polymorphonuclear leukocytes and monocytes (PMNms) ingesting fluorescently labeled, ethanol-fixed N. meningitidis organisms (phagocytic activity) in the presence of human sera was measured to reflect the serum opsonic activity against the bacterium. The contribution to opsonophagocytic activity of antibodies to inner core LPS was estimated by comparing the opsonic activities of adult and infant sera before and after adsorbing anti-LPS antibodies from the sera using purified LPS extracted from an LPS mutant (galE) of N. meningitidis strain MC58 (B:15:P1.7,16:L3). The specificity of the assay was further investigated using monoclonal antibody (MAb) B5, which binds to an inner core LPS epitope of N. meningitidis. A dose-dependent decrease in phagocytic activity was observed when MAb B5 was incubated with LPS from an inner core LPS (galE) mutant. Similarly, the number of PMNms ingesting fluorescently labeled polystyrene beads coated with inner core (galE) LPS decreased in a dose-dependent fashion when MAb B5 was incubated with various concentrations of the homologous inner core LPS. Strong correlations were found between the concentration of serum antibodies to inner core LPS (galE) versus the phagocytic activity using healthy adult sera (r(2) = 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r(2) = 0.99) using polystyrene beads coated with inner core LPS and opsonized with the same sera using the oxidative burst indicator system dihydrorhodamine123/rhodamine 123. OPA results were also found to correlate closely with the results of the serum bactericidal assay using MAb B5 against the N. meningitidis MC58 galE mutant in the presence of human complement (r(2) = 0.994, P = 0.003, two-tailed test). These studies demonstrate that functional antibodies are produced in humans against meningococcal inner core LPS and that the OPA is a useful approach to study the opsonic activity of antibodies to inner core LPS in health and disease.

Meniscal repair devices.

Meniscal repair devices not requiring accessory incisions are attractive. Many factors contribute to their clinical effectiveness including their biomechanical characteristics. This study compared several new meniscal repair devices with standard meniscal suture techniques. Using a porcine model, axis-of-insertion loads were applied to various meniscal sutures and repair devices. A single device or stitch was placed in a created meniscal tear and a load applied. Both loads and modes of failure were recorded. The load-to-failure data show stratification into 4 distinct statistical groups. Group A, 113 N for a double vertical stitch; group B, 80 N for a single vertical stitch; group C, 57 N for the BioStinger, 56 N for a horizontal mattress stitch, and 50 N for the T-Fix stitch; and group D, 33 N for the Meniscus Arrow (inserted by hand or gun), 32 N for the Clearfix screw, 31 N for the SDsorb staple, 30 N for the Mitek meniscal repair system, and 27 N for the Biomet staple. The failure mechanism varied. Sutures broke away from the knot. The Meniscus Arrow and BioStinger pulled through the inner rim with the crossbar intact. The Clearfix screw failed by multiple mechanisms, whereas 1 leg of the SDsorb staple always pulled out of the outer rim. The Mitek device usually failed by pullout from the inner rim. The Biomet staple always broke at the crosshead or just below it. Although the surgeon should be aware of the material properties of the repair technique chosen for a meniscal repair, this information is only an indication of device performance and may not correlate with clinical healing results.

Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the inner core lipopolysaccharide of Neisseria meningitidis group B.

We have developed a solid-phase ELISA to study the human immune response to inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm) using structurally defined glycolipids from a genetically defined mutant (galE) of a serogroup B Nm strain. Previous studies had demonstrated that a galE (inner core) LPS epitope is conserved in approximately 70% Nm strains and was accessible to antibody in fully encapsulated wild-type Nm strains. A murine monoclonal antibody, MAb B5, raised to a galE mutant of serogroup B Nm strain, immunotype L3 (B.15.P1.7,16) was used to determine the specificity of the inner core LPS ELISA by inhibition studies using purified galE LPS and human sera. The intra-assay coefficient of variation (CV) was 5-6% and inter-assay CV was 19-22%. Using this ELISA, significant differences in the geometric mean titres (GMTs) of naturally occurring serum antibodies (specific to inner core LPS) between healthy adults (18-65 years, N=54) and healthy infants (3-4 months, N=144) of both IgG and IgM classes were found (P<0.0001). GMTs were expressed in galE arbitrary units (AU/ml) (95% confidence intervals): IgG antibodies in adults 5.7 (5. 0,6.9) and in infants 1.1 (1.0,1.3); IgM antibodies in adults 7.7 (5. 7,10.4), and in infants 0.85 (0.7,1.1). In age-matched children aged 26-113 months a difference (P=0.04) in specific IgG was found in healthy infants and infants in the acute phase of invasive Nm disease (GMT (95%CI) in AU/ml: in healthy infants 7.7 (5.3,11.0), in acute phase infants 4.2 (2.5,7.2). However, there was no difference in specific IgM (P=0.98) between these groups healthy infants 4.7 (3. 1,7.0), acute phase 4.6 (2.9, 7.4). In eleven children (5-181 months) there were differences in the GMTs of specific IgG and IgM (P=0.02, P=0.008 respectively) between paired acute and convalescent sera (GMT) (95%CI) in AU/ml: IgG acute 1.95 (0.98, 3.8), convalescent 5.2 (2.2,12.4); IgM acute 1.78 (1.05,3.0), convalescent 4.38 (2.6,7.3). We conclude that ELISA is a specific, sensitive and reproducible method for the detection of antibodies to inner core LPS of Nm and that an epitope defined by MAb B5 can be immunogenic in infants and adults. These findings are relevant to the potential candidacy of inner core LPS as a vaccine.

Suture anchors--update 1999.

New suture anchors continue to become available. Our prior reports on the pullout strength of over 50 different anchors is supplemented by a similar test conducted on 25 additional new anchors. This anchor comparison, using an established protocol in fresh porcine femurs, recorded failure strength, failure mode (anchor pullout, suture eyelet cutout, or wire breakage), eyelet size, minor and major diameters, and drill hole sizes. These new anchors were tested in diaphyseal cortex, metaphyseal cortex, and a cancellous trough. Tensile stress parallel to the axis of insertion was applied at a rate of 12.5 mm/sec by an Instron 1321 until failure and mean anchor failure strengths were calculated. Anchors tested included DePuy 4.5 prototypes D1, D2 Catera 4.5, and D3; DePuy 3.5 prototypes D4- Catera 3.5, D5, and D6; Mainstay 2.7, 3.5, 4.5; ROC EZ 2.8, EZ 3.5, and XS 3.5; Ultrafix RC and Ultrafix MiniMite; 1.3 MicroMitek, Panalok 3.5, and Tacit 2.0; Umbrella Harpoon; PeBA 2.8, 4.0, 6.5; and Stryker 1.9, 2.7, 3.4, and 4.5 prototypes. Screw anchors still tend to have higher values, but for the newer nonscrew designs this distinction is less apparent. The new biodegradable anchors were all composed of poly L-lactic acid suggesting a trend away from other polymers, and these new biodegradable anchors showed load-to-failure strengths comparable to others in their class. All anchors were stronger than the suture for which they are designed to accommodate.

Kidney cografts enhance fiber outgrowth from ventral mesencephalic grafts to the 6-OHDA-lesioned striatum, and improve behavioral recovery.

Recent studies have demonstrated the presence of many different neurotrophic factors in the developing and adult kidney. Due to its production of this mixture of neurotrophic factors, we wanted to investigate whether fetal kidney tissue could be beneficial for neuritic fiber growth and/or cell survival in intracranial transplants of fetal ventral mesencephalic tissue (VM). A retrograde lesion of nigral dopaminergic neurons was performed in adult Fischer 344 male rats by injecting 6-hydroxydopamine into the medial forebrain. The animals were monitored for spontaneous locomotor activity in addition to apomorphine-induced rotations once a week. Four weeks following the lesion, animals were anesthetized and embryonic day 14 VM tissue from rat fetuses was implanted stereotaxically into the dorsal striatum. One group of animals received a cograft of kidney tissue from the same embryos in the same needle track. The animals were then monitored behaviorally for an additional 4 months. There was a significant improvement in both spontaneous locomotor activity (distance traveled) and apomorphine-induced rotations with both single VM grafts and VM-kidney cografts, with the VM-kidney double grafts enhancing the motor behaviors to a significantly greater degree. Tyrosine hydroxylase (TH) immunohistochemistry and image analysis revealed a significantly denser innervation of the host striatum from the VM-kidney cografts than from the single VM grafts. TH-positive neurons were also significantly larger in the cografts compared to the single VM grafts. In addition to the dense TH-immunoreactive innervation, the kidney portion of cografts contained a rich cholinergic innervation, as evidenced from antibodies against choline acetyltransferase (ChAT). The striatal cholinergic cell bodies surrounding the VM-kidney cografts were enlarged and had a slightly higher staining density for ChAT. Taken together, these data support the hypothesis that neurotrophic factors secreted from fetal kidney grafts stimulated both TH-positive neurons in the VM cografts and cholinergic neurons in the host striatum. Thus, these factors may be combined for treatment of degenerative diseases involving both dopaminergic and cholinergic neurons.

Molecular Methods for Haemophilus influenzae.

The speciesHaemophilus influenzae belongs to the genus Haemophilus and the family Pasteurellaceae. H influenzae are small, nonmotile, nonspore forming, Gram-negative, pleomorphic rods that range in shape from coccobacilli to long filaments. They require X and V factors (hemin and NAD, respectively) for aerobic growth, and may be facultatively anaerobic (1) Encapsulated H. influenzae are classified into six antigenically distinct serotypes (a-f), and have a clonal population structure with two major global subdivisions (I and II) (2,3). Nonencapsulated H. infuenzae (NCHi) appear to have a nonclonal population composition, but a broader analysis of NCHi in the future may reveal that the population is not so distinct from encapsulated H influenzae (4). Both encapsulated H influenzae and NCHi exhibit wide genetic diversity (5).

Internal fixation strength of suture anchors--update 1997.

Two new areas of anchor development are biodegradable anchors and "mini" anchors. The group of biodegradable anchors tested include the Bio-Anchor, LactoSorb, Biofix, Bio-Statak, Mini Screw suture anchor, DePuy 4.5 molded, DePuy 4.5 machined, DePuy 3.5 machined, TAG Wedge 4, TAG Rod 2, TAG Wedge 3, TAG Wedge 2, and Stealth. "Mini anchors" have drill holes or minor diameters of < 2.2 mm. Those tested include the Mini Revo and Bio-Anchor, miniHarpoon, mini Mitek and Fast in 3, Statak 1.5 and 2.5, SB 2 and PeBA 3, Corkscrew 5, Corkscrew 3.5, and Fastak A2, Ogden 2.5, TAG Wedge 2, ROC 1.9, and Questus 2.5. Additional anchors tested that fit neither category include the Anspach, Questus 3.5 and 5.0, SB 3 and PeBA-C, Ogden 3.5, Fast in 4, Ultrafix, and the ROC 3.5, ROC 2.8, ROC 2.3, and ROC XS. An anchor comparison, using an established protocol in fresh porcine femurs, recorded failure strength, failure mode, eyelet size, minor and major diameters, and drill hole sizes. Except for the Bio-Anchor and TAG Wedge 2, biodegradable anchors tend to be larger to compensate for their lower strength relative to metal. Biodegradable screw anchors' predominant failure mode was eyelet cutout, whereas biodegradable nonscrew anchors failed to predominantly by anchor pullout. From an initial mechanical perspective, these biodegradable anchors perform acceptably. Both biodegradable and "mini" anchors include screw and nonscrew designs. As expected, screw designs perform well and generally fail at higher loads than nonscrew anchors. Although biodegradable anchors, as a group, are not as strong as metal anchors, they are stronger than the sutures for which they are designed. The move to smaller ("mini") and biodegradable anchors is supported by these data. Whether an anchor fails at twice the suture breaking strength or 10 times the suture breaking strength should make no difference.

Effect of capacitive coupled electrical stimulation on regenerate bone.

An in vivo study was carried out to determine if capacitive coupled electrical stimulation increased the rate of recovery of strength of regenerate bone produced as a result of lengthening by the Ilizarov technique. Thirty-four adult male beagles underwent a right tibial mid-diaphyseal corticotomy, followed by a 5-day delay, and then 21 days of lengthening (1 mm/day). At the start of the post-distraction period (day 27), stimulation (3-6.3 V peak to peak, 5-10 mA root-mean-square at 60 kHz) was applied for 28 days to one group. The nonstimulated group (n = 17) underwent a 28-day period with no stimulation. From each group, four tibiae were prepared for histology; both ends of the remaining bones were embedded in polymethylmethacrylate and tested in torsion (internal rotation at 4.7 degrees/sec) until failure. Statistically significant changes included a 37% lower maximum torque capacity and a 40% decrease in strain energy to failure in the stimulated group compared with the nonstimulated group. The findings are supported by measured trends to a lower modulus of rigidity (37% decrease) and a smaller percentage of active osteoid perimeter (20% decrease) for the stimulated group. The experimental data suggest that when this dose of capacitive coupled electrical stimulation is applied to the regenerating bone created during distraction osteogenesis, it delays the recovery of bone strength compared with an untreated control.

Suture anchor strength revisited.

The rapid proliferation of suture anchors continues. Our prior report on the pullout strength of 14 different anchors is supplemented by a similar test conducted on 8 additional anchors. Comparative data on modes of failure and failure strengths (ultimate loads to failure) for these new devices are compared statistically with the previously tested anchors. In a fresh never-frozen porcine femur model, 10 samples of each of the additional anchors tested were threaded with stainless steel sutures and inserted into three different test areas (diaphyseal cortex, metaphyseal cortex, and a cancellous trough). Tensile stress parallel to the axis of insertion was applied at a rate of 12.5 mm/s by an Instron 1321 testing machine (Instron Corp, Canton, MA) until failure and mean anchor failure strengths calculated. The anchors tested were the Mitek G2 as a control, miniMitek, Mitek Superanchor, Mitek Rotator Cuff anchor (Mitek Products, Westwood, MA), Innovasive Devices Radial Osteal Compression device (Innovasive Devices, Hopkinton, MA), Arthrex Fastak (Arthrex Inc, Naples, FL), Arthrotek miniHarpoon (Arthrotek, Warsaw, IN), Orthopedic Biosystems PeBA 3 and PeBA 5 (Orthopedic Biosystems, Scottsdale, AZ), and AME 5.5 screw (American Medical Electronics, Richardson, TX). Failure mode (anchor pullout, suture eyelet cut out, or wire breakage) was generally consistent for each anchor type. The size of insertion hole is clinically important and each anchor's performance was evaluated as a function of its minor diameter or drill hole. For screw anchors, the larger the minor diameter of the screw, the higher the mean failure strengths in all three test areas (P = .001). However, larger drill holes for non-screw anchors resulted in lower mean failure strengths in cancellous bone (P = .03) and diaphyseal cortex (P < .005).

Meningococcal vaccines for the United Kingdom.

New meningococcal vaccines are needed in the United Kingdom with some urgency. Almost all Neisseria meningitidis disease in this country is caused by serogroups B and C. Infants have the highest attack rates, but also make the poorest immunological responses to potential vaccines. The development of vaccines that protect infants is a significant challenge. A capsule-based serogroup B vaccine is unlikely to be successful in infants because the capsule is poorly immunogenic and the polysaccharide molecule mimics a human epitope. Without completely discounting capsule as an immunogen, alternate antigens are being considered for immunisation: outer membrane proteins (OMP), iron regulating proteins, and lipopolysaccharide. Vaccines based on OMP have been used in several phase 3 trials in South Africa, Cuba, Brazil, Norway, and Chile, in which two doses of vaccine were given. The Cuban and Norwegian vaccines have been compared in phase 2 trials in Iceland and Chile. Potential limitations are epitope heterogeneity and the theoretical ability of N. meningitidis to adapt even to hosts who have received polyvalent vaccines. A phase 2 trial of a hexavalent class 1 OMP vaccine is under way in Gloucester, with 100 babies receiving injections at 2, 3, and 4 months. Serogroup C vaccines have been developed from capsular polysaccharide but, unconjugated, these vaccines do not protect those under 2 years of age. Conjugate vaccines with C and AC polysaccharides are immunogenic in infants, but antibody titres may wane quickly.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultimate strength of suture anchors.

Suture anchors of various designs are gaining acceptance for open and arthroscopic procedures. The rapid proliferation of these devices challenges those using them to apply objective criteria for device selection. Comparative data on implant security in different settings, modes of failure, and ultimate failure strengths is lacking. This study was undertaken to independently develop such data for an objective comparison of the suture anchors currently available. Using a fresh never-frozen porcine femur model, 10 samples of each of the 14 different anchors tested were inserted into each of the three different test areas; diaphyseal cortex (usually 3- to 4-mm thick), metaphyseal cortex (usually 1- to 2-mm thick), and a cancellous bone "trough". The suture anchors were threaded with 0.018-inch stainless steel wire or, for anchors requiring a more flexible suture, 0.018-inch stainless steel 1 x 7 wire braid. Tensile stress parallel to the axis of insertion was applied at a rate of 12.5 mm/second by an Instron 1321 (Instron Corp, City, State) until failure. Average failure strength was calculated for each anchor at each test area. The anchors tested were the Mitek G2, Mitek G3, Mitek G4 (Mitek Surgical Products, Norwood, MA), Linvatec Revo screw (Linvatec, Largo, FL), Acufex TAG Wedge, Acufex TAG Rod 2 (Acufex Microsurgical, Mansfield, MA), Statak models 1.5, 2.5, 3.5, 5.0, and 5.2 (Zimmer, Warsaw, IN), Arthrex ESP (Arthrex Inc., Naples, FL), Arthrotek Harpoon, and Arthrotek LactoSorb (Arthrotek, Warsaw, IN). The average failure strength of each of these anchors in the diaphyseal cortex, metaphyseal cortex, and cancellous bone is reported.(ABSTRACT TRUNCATED AT 250 WORDS)

Secondary amyloidosis from long-standing bacterial endocarditis.

Survival of patients with increasingly complex congenital heart disease has produced a population of children and adolescents who are susceptible to subacute bacterial endocarditis (SBE). We report a child whose endocarditis went unrecognised, and who developed amyloidosis. Asymptomatic proteinuria, haematuria and renal impairment are occasionally seen in SBE and usually indicate glomerulonephritis. Amyloidosis should also be suspected in children with long-standing bacterial endocarditis with proteinuria or other evidence of renal impairment, especially if associated with organomegaly. The diagnosis is made by renal biopsy.

Phytanic acid alpha-oxidase deficiency (Refsum disease) presenting in infancy.

This report describes a patient with high serum phytanic acid concentration due to phytanic acid alpha-oxidase deficiency (classical Refsum disease). He presented unusually early, hypotonia and developmental delay being apparent by 7 months. A generalized peroxisomal disorder (so-called 'infantile Refsum disease') was excluded by analyses of pristanic acid, very long-chain fatty acids, bile acids and plasmalogen synthesis. The early presentation raises the possibility of in utero exposure to phytanate.

Morphologic and histochemical characteristics of skeletal muscle after long-term intramuscular electrical stimulation.

The purpose of this investigation was to examine the morphologic and histochemical characteristics of paraspinal muscles in patients with scoliosis after long-term electrical stimulation. Thirty-six children with idiopathic scoliosis, who had been treated with implantable muscle stimulators, had paraspinal muscle biopsies at the time of implantable muscle stimulator removal. Group A patients whose curve did not progress, had 2.9 years of stimulation stopped at skeletal maturity, with a further 1.5 years of nonstimulation before implant removal and biopsy. In group B patients, who had an average of 2.3 years of stimulation, the curve progressed and stimulation was continued until fusion and biopsy. Neither group showed any increase in the frequency of pathologic changes of paraspinal muscles contrasted with values reported in the literature for scoliotic muscle. In group A patients there was an increased proportion of type 1 fibers on the convex side of the curve compared to the concavity. Despite this finding the curves did not require fusion, suggesting that the increased percentage of type 1 fibers was not the cause of the scoliosis. In group B patients there was an even higher type 1 concentration on the convex side contrasted to the convex side of group A patients.

Scoliosis treatment in children using a programmable, totally implantable muscle stimulator (ESI).

More than ten years ago, we began the night time treatment of early scoliosis in growing children with implanted muscle stimulators [1], [2]. The early devices were radio-frequency (RF) coupled units with an implanted receiver and external transmitter-antenna which the patient used at night to power and activate the implant. Compliance with this treatment was 95 percent. Recent developments have led to the use of a new, totally implantable stimulator for the treatment of single scoliotic curves. The unit has no external components, is programmed and interrogated by telemetry, and is externally switched by the patient using a magnet. Compliance with it continues at a high level, product reliability to date has been perfect, and the clinical results continue good.

Paraspinal muscle stimulation for the treatment of idiopathic scoliosis in children.

More than 12 years ago, we began to investigate the potential use of electrical stimulation of the paraspinal muscles to control scoliosis in children. Animal experimentation showed that unilateral stimulation of these muscles will cause the spine to grow toward the stimulated side. It was also shown that the stimulation need only be carried on for part of the day. We have developed a treatment for scoliosis based on night-time use of stimulation either with an implantable or surface system. Both single and dual channel systems are available for the treatment of single or double scoliotic curves. Treatment is carried on at night while the child is asleep, and there are no exercises or brace programs associated with it. Approximately 75% of the curves are either improved significantly, or maintained at their starting value. Only about 15% of the curves fail to respond and require a spinal fusion to stabilize them. Patient acceptance and compliance has been excellent.

Effect of low level direct current on in vivo tumor growth in hamsters.

A preliminary study has been carried out on the effect of low level direct current on tumor growth using an experimental tumor model developed from an amelanotic melanoma (T1-4) in the hamster. An inoculum of 2 X 10(6) viable cells was injected s.c. on day 0; on day 7 the tumor-bearing animals were randomly divided into treatment and control groups. On days 7 through 11 inclusive, the treatment group was subjected to electrical current (direct current) at levels from 0.1 to 2.4 mA, for 1 h/day under general anesthesia. Control groups were subjected to the same procedures, with the exception that the electrodes were not connected to the current source. On day 14, the animals were killed and autopsied; their tumors were removed, weighed, and sectioned. Treated tumors decreased in mass (as a percentage of controls) from 89% at 0.1 mA to 2% at 2.4 mA. Increased necrosis of the treated tumors was noted macroscopically and microscopically. On histological examination, it was observed that a thin rim of viable cells remained around the periphery after treatment even at the highest current levels. Similar results were obtained with both stainless steel and platinum-30% iridium electrodes. In separate experiments where the animals were allowed to survive after a treatment period (1 h/day for 5 days at 2.4 mA), the viable cells at the periphery developed into tumors whose mass at 28 days posttreatment averaged only 52% of that of the control tumors. The mechanism of growth reduction is unknown but hyperthermia was shown not to be a factor.