Production of a Sindbis/Eastern Equine Encephalitis chimeric virus inactivated cell culture antigen.

Eastern Equine Encephalitis virus (EEEV) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. Unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate EEEV infection from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the EEEV immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). However, EEEV is classified as a HHS select agent and requires biosafety level (BSL) three containment, limiting EEEV antigen production in non-select agent and BSL-2 laboratories. A recombinant Sindbis virus (SINV)/EEEV has been constructed for use under BSL-2 conditions and is not regulated as a select agent. Cell culture production of inactivated EEEV antigen from SINV/EEEV for use in the EEEV MAC-ELISA is reported here. Cell culture conditions and inactivation procedures were analyzed for SINV/EEEV using a recently developed antigen production algorithm, with the MAC-ELISA as the performance indicator.

Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Morphological adaptation of the bone graft and fused bodies after non-instrumented anterior interbody fusion of the lower cervical spine.

PURPOSE: To assess the remodelling process of the bone graft and fused bodies after non-instrumented anterior interbody fusion with autogenous iliac graft in patients with spondylosis, infections, fractures, or disorders of the cervical spine. METHODS: 68 patients aged 18 to 58 years who underwent non-plated anterior lower cervical interbody fusion with an iliac graft were retrospectively studied. Diagnoses of the patients were degenerative disc diseases (n=32), disc herniation (n=15), fractures (n=13), and tuberculosis (n=8). The Robinson and Smith technique was used to treat degenerative disc diseases and protruded disc, and the Bailey and Badgley procedure for fractures or tuberculosis of the cervical spine. 34, 25, and 9 patients underwent one-, 2-, and 3-segment fusions, respectively. 18 of the 25 patients underwent two-segment fusion with a single large bone block, and 7 with 2 separate bone blocks for each segment. Four of the 9 patients underwent three-segment fusion with a single large bone block, and 5 used separate grafts for each segment independently. Plain and stress radiography was primarily used to assess the fusion. Computed tomography and magnetic resonance imaging were also used in some patients. Some anterior graft extrusion (amounting to less than 10% of corresponding anteroposterior body width) was used to observe the remodelling during graft-take and thereafter. Postoperative cervical traction for 2 to 4 weeks, then cervical collar immobilisation for 4 to 12 weeks were strictly followed according to the numbers of fused segments. A halo vest was applied in 4 patients with fracture undergoing 3-segment fusion as they could not tolerate the prolonged bed rest or rigid cervical brace. RESULTS: The mean time for the graft to fuse was 8.6 (range, 7-14) weeks in patients who underwent each segment fusion with independent free grafts, and 10 and 14 weeks in those who underwent 2- and 3-segment single large graft fusion, respectively. The final loss of disc height and joint angle were negligible, regardless of the extent of fusion. Bony absorption of the anteriorly protruded part of the graft began at postoperative week 10 (range, 6-28), which coincided with the time of graft-take and initiation of remodelling. CONCLUSION: The earliest sign of bony absorption of the anteriorly protruded part of the graft indicated the initiation of the graft-take and the graft remodelling. The inwaisting sign of the surgically fused block of vertebral bodies was a morphological adaptation. Despite the altered biomechanics of the spine in the fused area, the inwaisting sign indicated maintenance of normal function at the parafusion motion segments.

Characterization of an extracellular dipeptidase from Streptococcus gordonii FSS2.

PepV, a dipeptidase found in culture fluids of Streptococcus gordonii FSS2, was purified and characterized, and its gene was cloned. PepV is a monomeric metalloenzyme of approximately 55 kDa that preferentially degrades hydrophobic dipeptides. The gene encodes a polypeptide of 467 amino acids, with a theoretical molecular mass of 51,114 Da and a calculated pI of 4.8. The S. gordonii PepV gene is homologous to the PepV gene family from Lactobacillus and Lactococcus spp.

Stabilisation of fractured thoracic and lumbar spine with Cotrel-Dubousset instrument.

OBJECTIVE: To evaluate the effectiveness of 2 different types of Cotrel-Dubousset instrument systems in stabilising thoracolumbar and lumbar spine fractures. METHODS: Between January 1989 and December 1993, 45 fractures in 42 patients with unstable fracture or fracture dislocation of the thoracolumbar and lumbar spines were randomly assigned to 2 surgical treatments with Cotrel-Dubousset instrumentation-using either a long segment (Group 1) or a short segment (Group 2)-and short posterolateral fusion. RESULTS: Consolidation of the fractured vertebral body and posterolateral fusion were achieved at a mean time of 4.5 months; fusion rates were 75% in Group 1 and 83% in Group 2. The average collapses of anterior vertebral body height in Group 1, in the immediate postoperative period and at the final follow-up, were 15% and 17%, respectively; and in Group 2, the figures were 16% and 24%, respectively. The correction of vertebral height and kyphosis at the last follow-up were lost more in Group 2 (5.7 degrees ) than in Group 1 (4.4 degrees ). There were neurological recoveries in 6 of the 9 cases of incomplete paraplegics, including complete recovery in 5, and one-Frankel grade increase in one. There were 15 instrument failures in 12 patients, including screw breakage in 3 Group 1 cases and 6 Group 2 cases. The plug dislodged in 3 Group 1 cases, and the hook dislodged in 3 Group 2 cases. In other words, instrument failures were more common in Group 2. CONCLUSION: Cotrel-Dubousset stabilisation of the fractured spine achieves fracture consolidation, but does not maintain the restored height and sagittal curve completely until fusion. The long rod and short fusion construct was more effective for all fracture types than was the short rod and fusion construct, although it leads to wider immobilisation of normal segments.

Brooks' posterior stabilisation surgery for atlantoaxial instability: review of 54 cases.

PURPOSE: To assess the effectiveness of Brooks' posterior stabilisation and fusion for the unstable atlantoaxial joint due to congenital dysplastic dens and trauma. METHODS: We retrospectively studied records of 54 patients (36 males and 18 females; age range, 3-58 years) who underwent Brooks' posterior stabilisation procedure between March 1975 and December 1999, at the Catholic University of Korea Medical Center and Dong-Shin General Hospital, Seoul. A single-stranded Kirschner wire was used to stabilise the first 19 cases (thin wires in 12 cases and thick wires in 7), and double-stranded wires were used in the next 35 cases (thin wires in 4 cases and thick wires in 31). After surgery, patients were immobilised in bed with light Halter traction of the head, followed by cervical bracing. RESULTS: Fusion was observed by X-ray postoperatively at 15 weeks in 48 patients. Reduction was achieved in 3 luxation cases (including the single case of rotatory fixation). Brooks' fusion failed in 4 patients with dens fractures and 2 with dens anomaly. Four dens fractures in cases of successful Brooks' fusion in Brooks' fusion did not unite. Wire failure occurred in 4 cases of thin single-stranded wire fixation, namely, 2 cases of dens fractures and 2 of dens anomaly. CONCLUSION: Brooks' procedure is safe and has a high fusion rate when double-stranded strong wire fixation of the atlantoaxial joint is combined with meticulous bone grafting and subsequent cervical bracing.

Isolation and characterization of a highly specific serine endopeptidase from an oral strain of Staphylococcus epidermidis.

Infection by Staphylococcus epidermidis, an opportunistic pathogen, has become a major problem due to the increased use of implanted medical devices and the growing number of patients who are therapeutically or infectiously immunosuppressed. These infections appear to proceed via modulation of the coagulation and complement systems. In this communication we describe the purification and characterization of a novel extracellular proteinase from an oral strain of S. epidermidis that can degrade fibrinogen, complement protein C5, and several other proteins. This proteinase has a strong preference for cleavage after glutamic acid residues, but not after aspartic acid. The S. epidermidis enzyme may be a multifunctional protein which not only provides this organism with both the ability to evade the complement defense system and to dysregulate the coagulation cascade, but also supplies nutrients for its growth through the degradation of Glu-rich proteins.

Traumatic spondylolisthesis of the axis: 42 cases.

Fourty-two patients (34 males and 8 female) with traumatic spondylolisthesis of the axis were studied in a retrospective review There were 20 stable and 22 unstable fractures. The 22 unstable fractures were treated surgically: 16 anterior interbody fusion (10 non-plated and 6 plated), 4 pedicle screw fixation for osteosynthesis of the fractured pedicles, and 2 posterior wire fixation for flexion and axial load injury. For all non-surgical cases, head halter tractions for 1 to 8 weeks was prescribed and a cervical orthosis was worn for an additional 6 to 18 weeks. The surgical cases underwent 5 to 7 days of preoperative and 1 to 4 weeks of post-operative head halter traction. In all cases pedicle fractures united after 13 weeks on average in group treated conservatively, 12 weeks (11 to 13 weeks) in the posterior wiring group, 8 weeks (7 to 9 weeks) in the group in which pedicle screws were used, and 11 weeks (9 to 15 weeks) in the anterior fusion group (13 weeks in non-plated, and 8 weeks in plated). There were no differences in patterns of anterior fusion between those in the non-plated and plated groups. There were no non-unions of fractured pedicles and there was no late instability of the C2-C3 or neurological complications. In 2 cases in the posterior surgery group, there was mild nuchal discomfort and some rigidity for a short while postoperatively. Final outcomes were good in all cases.

Expression of brain/kidney protein in Müller cells of rat retina following transient ischemia.

We have investigated the expression and cellular localization of brain/kidney (B/K) protein in the rat retina following transient ischemia. In the normal retina, strong B/K immunoreactivity was localized to some ganglion cells. In addition, a few radial Muller cell processes showed B/K immunoreactivity. Following ischemia and reperfusion, most B/K-labeled ganglion cells were lost, whereas between 1 day and 2 weeks post-lesion B/K immunoreactivity appeared in many more Muller cell processes with increasing intensity. Quantitative evaluation by immunoblotting confirmed that B/K expression then decreased progressively, to 35% of control values at four weeks post-lesion. Our findings suggest that Muller cells are involved in the pathophysiology of retinal ischemia through the expression of B/K following transient ischemia.

Pott's Paraplegia--67 cases.

Sixty-seven patients were treated for Pott's paraplegia: 58 were adults and 9 were children. Sixty-four patients had active disease, and 3 had healed disease. All patients had triple chemotherapy with or without decompression surgery. Thirteen patients, including 9 children, were treated conservatively, whereas 54 patients who met the selection criteria for surgery were treated surgically. Fifty-two patients had anterior radical decompression surgery, and for 14 of them, anterior surgery was preceded by posterior instrumental stabilization surgery. Two patients with healed disease had posterior decompressive corpectomy. There was functional recovery in 60 (89.6%) patients, including 13 who had active disease that was treated conservatively. In 47 of the 54 surgically treated patients there was neurologic recovery, and 2 of these recovered incompletely with some residual spasticity. In the remaining 7 patients, there was no recovery. It took 2 to 6 months for recovery for the patients with conservative treatment, whereas it took <2 months for the patients with anterior decompression. The patients who had the combined 2-stage procedure could be mobilized earlier after neurologic recovery than could the patients having the anterior radical surgery and the conservatively treated patients. It was proven that paraplegia of active disease can be treated successfully by conservative or surgical means and that paraplegia caused by healing of fibrosis in the severely deformed spine was difficult to treat successfully, even with radical surgery.

Breast engorgement: contributing variables and variables amenable to nursing intervention.

The focus of this study was to identify variables that correlate significantly with breast engorgement and that might be amenable to nursing interventions. Data on the initiation of feeding, frequency of feedings, feeding duration, rate of milk maturation, and supplementation were obtained of 54 women. These variables were found to be significantly correlated with breast engorgement.