Cloning and expression of Trypanosoma cruzi ribosomal protein P0 and epitope analysis of anti-P0 autoantibodies in Chagas' disease patients.

Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a major cause of heart failure in endemic areas. Antigenic mimicry by T. cruzi antigens sharing epitopes with host macromolecules has been implicated in the pathogenesis which is thought to have a significant autoimmune component. We report herein on the cloning and characterization of a full-length cDNA from a T. cruzi expression library encoding a protein, TcP0, that is homologous to the human 38-kD ribosomal phosphoprotein HuP0. The T. cruzi P0 protein shows a clustering of residues that are evolutionarily conserved in higher eukaryotes. This includes an alanine- and glycine-rich region adjacent to a highly charged COOH terminus. This "hallmark" domain is the basis of the crossreactivity of the highly immunogenic eukaryotic P protein family. We found that T. cruzi-infected individuals have antibodies reacting with host (self) P proteins, as well as with recombinant TcP0. Deletion of the six carboxy-terminal amino acids abolished the reactivity of the T. cruzi infection sera with TcP0. This is similar to the specificity of anti-P autoantibodies described for a subset of patients with systemic lupus erythematosus (SLE) (Elkon, K., E. Bonfa, R. Llovet, W. Danho, H. Weissbach, and N. Brot. 1988. Proc. Natl. Acad. Sci. USA. 85:5186). These results suggest that T. cruzi P proteins may contribute to the development of autoreactive antibodies in Chagas' disease, and that the underlying mechanisms of anti-P autoantibody may be similar in Chagas' and SLE patients. This study represents the first definitive report of the cloning of a full-length T. cruzi antigen that mimics a characterized host homologue in structure, function, and shared antigenicity.

A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin 12.

Leishmania braziliensis causes cutaneous and mucosal leishmaniasis in humans. Most patients with cutaneous leishmaniasis heal spontaneously and may therefore have developed protective immunity. There appears to be a mixed cytokine profile associated with active cutaneous or mucosal disease, and a dominant T helper (Th)1-type response associated with healing. Leishmanial antigens that elicit these potent proliferative and cytokine responses from peripheral blood mononuclear cells (PBMC) are now being identified. Herein, we report on the cloning and expression of a L. braziliensis gene homologous to the eukaryotic ribosomal protein eIF4A (LeIF) and patient PBMC responses to rLeIF. Patients with mucosal and self-healing cutaneous disease had significantly higher proliferative responses than those with cutaneous lesions. Whereas the parasite lysate stimulated patient PBMC to produce a mixed Th1/Th2-type cytokine profile, LeIF stimulated the production of interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha but not IL-4 or IL-10. Recombinant LeIF (rLeIF) downregulated both IL-10 mRNA in the "resting" PBMC of leishmaniasis patients and LPS-induced IL-10 production by patient PBMC. rLeIF also stimulated the production of IL-12 in cultured PBMC from both patients and uninfected individuals. The production of IFN-gamma by patient PBMC stimulated with either rLeIF or parasite lysate was IL-12 dependent, whereas anti-IFN-gamma monoclonal antibody only partially blocked the LeIF-induced production of IL-12. In vitro production of both IFN-gamma and IL-12 was abrogated by exogenous human recombinant IL-10. Therefore, we have identified a recombinant leishmanial antigen that elicits IL-12 production and Th1-type responses in patients as well as IL-12 production in normal human PBMC.

Diminished exoproteome of Frankia spp. in culture and symbiosis.

Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N(2) fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

Probing the structure and mechanism of Ras protein with an expanded genetic code.

Mutations in Ras protein at positions Gly12 and Gly13 (phosphate-binding loop L1) and at positions Ala59, Gly60, and Gln61 (loop L4) are commonly associated with oncogenic activation. The structural and catalytic roles of these residues were probed with a series of unnatural amino acids that have unusual main chain conformations, hydrogen bonding abilities, and steric features. The properties of wild-type and transforming Ras proteins previously thought to be uniquely associated with the structure of a single amino acid at these positions were retained by mutants that contained a variety of unnatural amino acids. This expanded set of functional mutants provides new insight into the role of loop L4 residues in switch function and suggests that loop L1 may participate in the activation of Ras protein by effector molecules.

Cell-free nitrogenase and hydrogenase from actinorhizal root nodules.

Field-grown alder (Alnus glutinosa) root nodules were disrupted in liquid nitrogen to release the actinomycete endophytes. The endophytes were broken by mild sonic oscillation and yielded a cell-free nitrogenase preparation capable of reducing acetylene and protons. In addition, the preparation carried a cell-free uptake hydrogenase.

Sequences of nifX, nifW, nifZ, nifB and two ORF in the Frankia nitrogen fixation gene cluster.

The actinomycete Frankia alni fixes N2 in root nodules of several non-leguminous plants. It is one of the few known N2-fixing members of the high-GC Gram+ lineage of prokaryotes. Thus, we have undertaken a study of its nitrogen fixation gene (nif) organization to compare with that of the more extensively characterized proteobacteria. A cosmid (pFN1) containing the nif region of Fa CpI1 was isolated from a cosmid library using the nifHDK genes of Fa CpI1 as a probe. A 4.5-kb BamHI fragment that mapped downstream from the previously characterized nifHDK genes was cloned and sequenced. Based on nt and aa sequence similarities to nif from other N2-fixing bacteria, eight ORF were identified and designated nifX, orf3, orf1, nifW, nifZ, nifB, orf2 and nifU. A region that hybridized to Rhizobium meliloti and Klebsiella pneumoniae nifA did not appear to contain a nifA-like gene. We have revised the map of the Fa nif region to reflect current information.

Early failure of short-segment pedicle instrumentation for thoracolumbar fractures. A preliminary report.

The results after treatment of fifty-two lumbar and thoracolumbar fractures with Cotrel-Dubousset instrumentation were reviewed as part of an ongoing study. Nineteen patients (average duration of follow-up, fifteen months) had been managed with short-segment pedicle-screw instrumentation. This preliminary report outlines the complications and pitfalls identified during the initial healing phase in this subgroup of patients. There were no neurological or vascular injuries due to placement of the pedicle screws, but ten patients had some form of failure of the fixation during the early period of healing. Failure of the fixation was manifested in three ways: progressive kyphosis secondary to the bending of screws (six patients), kyphosis secondary to osseous collapse or vertebral translation without bending of the hardware (three patients), and segmental kyphosis after a caudad screw in the lumbar construct broke (one patient, who had had a combined instrumentation for multiple fractures). Untreated anterior instability, and pre-stressing of the screws when the rods were contoured in situ, resulted in a high rate of failure. The high rate of failure of the hardware associated with this fixation construct suggests that posterior screw fixation alone may not be adequate when Cotrel-Dubousset instrumentation is used for short-segment lumbar arthrodeses. Bent screws or measurable kyphosis did not always herald a clinical failure, but patients who had progressive kyphosis of more than 10 degrees had substantially more pain than did those who had little or no progression. The results reported here are preliminary, and speculation as to the importance of these findings and as to the long-term outcome in these patients would be premature.(ABSTRACT TRUNCATED AT 250 WORDS)

The spine in osteogenesis imperfecta.

A high incidence of spinal curvature, approaching 80 per cent in patients more than twelve years old, was found in a large series of patients with osteogenesis imperfecta. Curves showed early progression and a predictable pattern. In the age range between one and five years the incidence of scoliosis was 26 per cent; thereafter, it rose precipitously. The degree of curvature was greater in the older children. The Milwaukee brace was ineffective in the treatment of the scoliosis and the complications of its use were detrimental. In several patients it deformed the rib cage. It had little or no effect on the progression of the curve. The incidence and degree of curvature was related to the type of bone present. Patients with chest deformities and those who were not ambulatory seemed to have a predilection for scoliosis.

Roentgenographic evaluation of vertebral rotation.

To examine the accuracy of vertebral rotation determinations made from roentgenographic pedicle-shadow offset measurements, roentgenograms were made of fifteen cadaver vertebrae with different sagittal and frontal inclinations and longitudinal axis rotations ranging from 15 degrees left to 45 degrees right. It was found that significant uncertainties arise when vertebral rotation is evaluated from measurements of pedicle-shadow offset: true and apparent offsets in a single vertebra differ markedly; vertebrae from different spines have different offsets for the same degree of rotation and the same inclinations; and changes in the sagittal and frontal inclinations of a single vertebra alter its offset in the same manner as changes in rotation. From these results, it seems unlikely that precise measurements of rotation changes in spines with mild or moderate scolioses, for example, can be made from anteroposterior roentgenograms.

Intraoperative autologous transfusion in orthopaedic patients.

The cases of 175 consecutive patients who had intraoperative autologous transfusion during revision total hip arthroplasty, an elective operation on the spine, repair of trauma to the spine, or open reduction of a fracture of the acetabulum were reviewed to evaluate the applicability of this technique in orthopaedic operations. A separate group of forty-one consecutive patients who had open reduction of a fracture of the acetabulum or the spine before the introduction of the autotransfuser was reviewed and compared with the group that had autotransfusion. An autologous blood predeposit program was used for twenty-five of fifty-two patients who had a procedure on the hip and for fifty-one of fifty-five patients who had an elective procedure on the spine. The mean rate of red blood-cell salvage using the autotransfuser was 60 per cent over-all. The mean transfusion requirements were significantly less (p less than 0.001) in all groups of patients in whom the autotransfuser was used. Use of the autotransfuser reduced the mean requirement for banked blood in patients who had a fracture of the acetabulum from 3.8 to 2.3 units per patient, and significantly reduced the mean need for banked blood in individuals who had trauma to the spine from 2.7 to 1.8 units per patient (p less than 0.01). The use of prebanked autologous blood further reduced the mean requirement for homologous blood from 2.4 to 0.8 unit per patient in those who had revision total hip arthroplasty (p less than 0.005), and from 3.6 to 0.4 unit per patient in those who had an elective procedure on the spine (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical properties of the tibia from chickens with idiopathic scoliosis.

The tibia from six-week old chickens that develop idiopathic scoliosis were studied with stress relaxation experiments and torsional strength testing. Most parameters observed did not show any significant differences between tibias obtained from chickens with scoliosis and tibias from the control birds; however, the rate of stress relaxation of the tibia from the birds with scoliosis was minimally increased over the controls. There were no significant differences noted in ultimate torsional strength, maximum angular deformity or modulae of torsional rigidity of the tibias from scoliotic chickens when compared to tibias from control chickens.

Urgent surgical stabilization of spinal fractures in polytrauma patients.

STUDY DESIGN: A prospective, longitudinal study of multiply injured patients treated with segmental instrumentation for spinal fractures with a minimum 2-year follow-up. OBJECTIVES: To determine whether urgent stabilization of spinal fractures in severely injured patients increases the risk of surgery compared with early treatment and historical results. SUMMARY AND BACKGROUND DATA: Opinion in clinical studies is divided about whether operative treatment offers an advantage over nonoperative treatment in isolated spine fractures. Concomitant trauma is rarely discussed relative to decision making or surgical timing. Urgent stabilization of long-bone fractures improves survival and outcome in polytrauma patients. To date, urgent treatment of spine fractures in polytrauma patients has not been considered in the literature. METHODS: Seventy-five consecutive patients treated with segmental instrumentation for spinal trauma were observed prospectively to assess perioperative and longterm outcome. Twenty-seven patients with severe polytrauma (injury severity score, > 26) were separately analyzed. Perioperative and postoperative results were analyzed relative to timing of surgery, injury severity score, and surgical approach. Urgent treatment was defined as that provided within 24 hours of the spinal injury, and early treatment was defined as that provided between 24 and 72 hours after injury. RESULTS: Twenty-five patients (93%) sustained two or more major injuries in addition to the spine fracture, and 17 of 27 (63%) had neurologic injury. The mean injury severity score approached or exceeded the LD50 (50% expected mortality) in each group--36.0 for the early-treatment group and 42.0 for the urgent group--but only one patient in each group died. There were no deep venous thromboses, pulmonary emboli, neurologic injuries, decubiti, deep wound infections, or episodes of sepsis in either group. Blood loss for anterior procedures was significantly higher in the urgent group, but estimated blood loss for posterior procedures was similar for both groups. At 49 months' mean follow-up, no revisions were necessitated by the urgent spinal treatment. CONCLUSIONS: Urgent spinal stabilization is safe and appropriate in polytrauma patients when progressive neurologic deficit, thoracoabdominal trauma, or fracture instability increase the risks of delayed treatment.

Neuromorphometric changes in the ventral spinal roots in a scoliotic animal.

The morphologic changes were investigated in the ventral and dorsal spinal nerve roots in a strain of white leghorn chickens that genetically was predisposed to have idiopathic scoliosis. In the ventral spinal roots in chickens with scoliotic curves, small myelinated fibers were increased in number and appeared in clusters. The number of small fibers did not correlate with the degree or the duration of the deformity. In 8 of the 15 chickens of the genetic scoliotic strain that did not have spinal curves, changes in the myelinated fibers, similar to those in chickens with scoliotic curves, were seen. These findings suggest that abnormalities in the myelinated fibers of the ventral spinal nerve roots may be the primary genetic lesion that, with variable penetrance, influences the development of scoliosis.

Intraoperative ultrasonography for reduction of thoracolumbar burst fractures.

The purpose of this study was to determine the efficacy of intraoperative ultrasound monitoring in the reduction and stabilization of thoracolumbar burst fractures of the spine. Thirty-one patients underwent a posterior approach for reduction and stabilization of a thoracolumbar burst fracture, with complete follow-up available on 26. Intraoperative real-time sonography was used for monitoring retropulsed fragments during distraction and fracture impaction. Computed tomography was used to compare preoperative canal compromise and postoperative reduction. Average canal compromise preoperatively was 66.5%, and the average canal compromise postoperatively was 18.7%. Neurologic function was not changed in 16, improved in ten, and worsened in none. Average hospital stay was 21.6 days. No complications were directly attributable to the use of the ultrasound. The authors believe that ultrasonography provides a safe and accurate method of intraoperatively evaluating reduction of burst fracture of the thoracolumbar spine from the posterior approach.

Sub-axial cervical dissociation. Anatomic and biomechanical principles of stabilization.

Four fresh human cadaver spines were analyzed during and after disruptive hyperflexion and hyperextension to characterize the pathoanatomy of three-column cervical dissociation. In both flexion and extension, the posterior longitudinal ligament and facet capsules provided the greatest resistance to disruption. At low loading rates, all structures failed through the soft tissues. Three-column disruption caused by either pure flexion or extension resulted in marked elongation of the neural axis, inconsistent with cord survival. Biomechanical studies were carried out in seven additional fresh frozen human cadaver specimens to determine the most rigid method of internally stabilizing three-column cervical dissociations. Specimens were tested in compressive flexion and distractive extension to evaluate stability of anterior, posterior, and combined fixation constructs. Specimens were tested intact, after partial two-column disruption, and after complete three-column dissociation. Posterior wiring provided significantly better flexural stability in two- rather than three-column disruptions. Posterior wiring reduced posterior displacement in two-column partial disruptions to 25% of control. In three-column dissociations, posterior wiring only reduced posterior displacement to 50% of control. In extension, posterior wiring was ineffective in preventing displacement. Anterior plating, used alone, tolerated only 37% of the maximum flexion moment before early failure. On the other hand, combined anterior plating and posterior Roger's wiring reduced posterior displacement in flexion to 20% of control, while reducing anterior displacement in extension to 50% of control. This improvement over the other constructs was statistically significant. In highly unstable cervical injuries, Morscher anterior cervical plates and modified Roger's posterior wire fixation provide a safe, rigid construct that protects neural function while allowing early and aggressive mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Iliac crest bone graft. Osteotome versus saw.

The strength of saw-harvested vs. osteotome-harvested Smith-Robinson iliac crest grafts from five fresh frozen cadavers was compared. Matched pairs of grafts were harvested, one with a saw and the other with an osteotome from equivalent locations on the two iliac crests of each pelvis. Grafts were tested to failure in axial compression, and load-displacement curves were recorded for each test. Yield load and displacement, ultimate load, and stiffness were calculated, and statistical analysis was performed with the Student t test and three-way analysis of variance. Evaluation of the 66 pairs of grafts revealed that saw-harvested grafts were consistently stronger than were matched grafts harvested with an osteotome. The middle third of the iliac crest produced significantly stronger grafts, and those harvested from younger donors were significantly stronger than those obtained from older donors. Of even greater clinical relevance, saw-harvested grafts were stiffer than osteotome-harvested grafts. Based on these findings, it is recommended that iliac crest grafts harvested for spine fusion be obtained with an oscillating saw rather than with an osteotome.

Biomechanics of cervical spine internal fixation.

Recent advances in the area of cervical spine internal fixation have resulted in important additions to the armamentarium of the spine surgery. However, a sophisticated knowledge of the biomechanics of these devices is important. This article discusses the biomechanics of odontoid screws, anterior cervical plates, posterior cervical plates, and posterior C1-C2 screw arthrodesis. It is hoped that this information will aid in implant selection.

The effect of anterior osteophytes and flexural position on thoracic trabecular strain.

STUDY DESIGN: Compressive and shear trabecular strains were evaluated using six cadaveric thoracic spines that included anterior osteophytes. The treatments were divided into three groups: 1) osteophytes intact and the specimen in the neutral position, 2) osteophytes removed and the specimen in the neutral position, and 3) osteophytes removed and the specimen with 5 degrees of additional flexion. OBJECTIVES: To investigate the influence of osteophytes and flexural position on vertebral trabecular strain during axial compression. SUMMARY OF BACKGROUND DATA: In the thoracic spine, the incidence of anterior wedge fractures increases with the severity of kyphosis. It is unclear whether the role of anterior osteophytes in the thoracic spine is to restrict progressive kyphosis, conduct axial load anteriorly, or both. METHODS: Thoracic motion segments, T10-T12, were axially loaded in compression, and the minimum principal and maximum shear strains were measured using texture correlation. RESULTS: No dramatic changes were found in the spatial distribution of the strains following removal of the anterior osteophytes. Conversely, after removal of the osteophytes and orienting the specimen in 5 degrees of additional flexion, the strain distribution shifted anteriorly and the magnitude increased. CONCLUSIONS: This study demonstrated that osteophytes seem to restrict progressive kyphosis rather than conduct axial load anteriorly.

The role of acute decompression and restoration of spinal alignment in the prevention of post-traumatic syringomyelia: case report and review of recent literature.

STUDY DESIGN: Case report. INTRODUCTION: Acute post-traumatic syringomyelia formation after spinal cord injury has been considered a rare complication. At this writing, most recent reports have surfaced in neurosurgical journals. As an entity, post-traumatic syringomyelia has not been widely appreciated. It has been confused with conditions such as Hansen's disease or ulnar nerve compression at the cubital tunnel. One study also demonstrated that the occurrence of syrinx is significantly correlated with spinal stenosis after treatment, and that an inadequate reduction of the spine may lead to the formation of syrinx. This reported case describes a patient in whom post-traumatic syringomyelia began to develop 3 weeks after injury, which improved neurologically after adequate decompression. SUMMARY OF BACKGROUND DATA: A 30-year-old man sustained a 20-foot fall at work. He presented with a complete spinal cord injury below T4 secondary to a T4 fracture dislocation. The patient underwent open reduction and internal fixation of T1-T8. After 3 weeks, the patient was noted to have ascending weakness in his bilateral upper extremities and some clawing of both hands. METHODS: A computed tomography myelogram demonstrated inability of contrast to pass through the T4-T5 region from a lumbar puncture. An incomplete reduction was noted. The canal showed significant stenosis. A magnetic resonance image of the patient's C-spine showed increased signal in the substance of the cord extending into the C1-C2 area. The patient returned to the operating room for T3-T5 decompressive laminectomy and posterolateral decompression including the pedicles, disc, and posterior aspect of the body. Intraoperative ultrasound monitoring showed a good flow of cerebrospinal fluid past the injured segment. RESULTS: On postoperative day 1, the clawing posture of the patient's hands was significantly diminished, and the patient noted an immediate improvement in his hand and arm strength. Over the next few days, the patient's strength in the bilateral upper extremities increased to motor Grade 4/5 on manual testing. A magnetic resonance image 4 weeks after decompression showed significant improvement in the cord diameter and signal. CONCLUSIONS: Post-traumatic syringomyelia has not been reported at so early a stage after injury. This disorder is an important clinical entity that must be recognized to prevent potentially fatal or devastating complications. As evidenced by the reported patient and the literature, if this disorder is discovered and treated early, permanent deficit can be avoided. The prevention of post-traumatic syringomyelia requires anatomic realignment and stabilization of the spine without stenosis, even in the case of complete injuries, to maintain the proper dynamics of cerebrospinal fluid flow.

Short-segment pedicle instrumentation. Biomechanical analysis of supplemental hook fixation.

STUDY DESIGN: This biomechanical study of fractures in cadaver vertebrae used specially designed pedicle screws to determine screw strains during loading of two different fixation constructs. OBJECTIVES: The authors determined the relative benefit of adding offset sublaminar hooks to standard pedicle screw constructs to reduce screw bending moments and prevent fixation failure and sagittal collapse. SUMMARY OF BACKGROUND DATA: Clinical studies have demonstrated a high incidence of early screw failure in short-segment pedicle instrumentation constructs used to treat unstable burst fractures. Strategies to prevent early construct failure include longer constructs, anterior strut graft reconstruction, and use of offset sublaminar hooks at the ends of standard short-segment pedicle instrumentation constructs. METHODS: Human cadaver spines with an L1 burst fracture were instrumented with a standard short-segment pedicle instrumentation construct using specially instrumented pedicle screws. Mechanical testing was carried out in flexion, extension, side bending, and torsion, and stiffness and screw bending moments were recorded. Offset hooks were applied initially, then removed and testing repeated. Stiffness data were compared to intact and postfracture results, and between augmented and standard constructs. RESULTS: Addition of offset laminar hooks, supralaminar at T11 and infralaminar at L2, to standard short-segment pedicle instrumentation constructs increased stiffness in flexion by 268%, in extension by 223%, in side bending by 161%, and in torsion by 155% (all were significant except torsion). Sublaminar hooks also reduced pedicle screw bending moments to roughly 50% of standard in both flexion and extension (P < 0.05). CONCLUSIONS: Supplemental offset hooks significantly increase construct stiffness without sacrificing principles of short-segment pedicle instrumentation, and absorb some part of the construct strain, thereby reducing pedicle screw bending moments and the likelihood of postyield deformation and clinical failure.