DNA target motifs of somatic mutagenesis in antibody genes.

During humoral immunity to T-cell-dependent antigens, responding B lymphocytes selectively mutate their antibody variable region genes at a high rate. This, together with the process of clonal selection, ultimately enhances the affinity and specificity of the antibody molecule and memory B cells that express it as a receptor. Despite several decades of investigation, the mutation mechanism has remained unresolved, largely due to the convoluted nature of experimental systems used to approach it. Somatic mutations preferentially occur within specific oligonudeotide motifs, and this targeting is consistent in all immunoglobulin genes of humans and mice that we have examined, suggesting that a conserved mechanism operates in both species. Our mutation targeting analyses implicate evolution of germline variable gene sequences to direct somatic mutations to specific codon positions in a manner that regulates the frequency of amino acid replacements to the benefit of the antibody product. Finally, our recent strand bias analyses support the idea that somatic mutation occurs preferentially, perhaps exclusively, at two bases on both strands of DNA. These and related observations from other laboratories support a mutation model that invokes at least two error-prone polymerases that have distinct template biases and requirements for elements of postreplicative mismatch repair.

Vascularity of the hip labrum: a cadaveric investigation.

PURPOSE: The purpose of this study was to define the vascularity of the hip labrum and to identify regional differences in vascular penetration that may have implications for the healing potential of this structure. TYPE OF STUDY: Injection study of human cadavers to investigate the vascularity of the hip labrum. METHODS: Twelve hips from 6 human frozen cadavers devoid of severe articular pathology were used. High-resolution surface-coil magnetic resonance imaging (MRI) was performed in both sagittal and coronal planes to better define anatomic planes in 2 dimensions and to correlate Spalteholz sections with the surrounding joint and labral anatomy. Each pelvis was injected with intra-arterial India Ink and frozen; 3-mm sagittal or coronal sections were then cut and processed using a modified Spalteholz technique, yielding anatomic zones of the labrum. Six specimens were cut in the sagittal plane and 6 were cut in the coronal plane. Specimens were examined at x10 magnification with transillumination. Anatomic zones with regional variations in vascularity were defined. RESULTS: The anterior and superior aspects of the labrum showed degenerative changes on MRI and under direct manual transillumination in 75% of specimens. Overall, there was a relatively poor vascular supply to the labrum; however, there were regional differences between anatomic zones. Zone I (capsular contribution) had significantly more vascularity than zone II (articular side) (P < .01). Zone IA (the portion of the zone not attached to bone) showed the most consistent source of vessels across all specimens (smallest variation between specimens); however, zone IB (the portion of the zone attached to bony acetabulum) had the greatest overall mean vascularity score. These differences were not statistically significant. Furthermore, vascularity patterns were not significantly different in the anterior, superior, posterior, and inferior labral regions, nor were they different in torn versus intact specimens. CONCLUSIONS: The cadaveric specimens evaluated in this study had a relatively avascular hip labrum. However, the increased vascularity seen in zone I (capsular side) may have implications for treatment, similar to that described in the meniscus of the knee. CLINICAL RELEVANCE: A better understanding of the vascularity of the hip labrum will guide treatment of labral pathology and may have implications for the healing potential of this structure. Labral tears occurring in the vascular zone may be amenable to arthroscopic repair rather than debridement.

Sequence-specific targeting of two bases on both DNA strands by the somatic hypermutation mechanism.

Somatic mutations within antibody genes alter the affinity and selectivity of antibody molecules and largely define the quality of the memory B cell repertoire in many vertebrate species. While some evidence supports the idea that there is a strand bias to the hypermutation mechanism, conflicting data suggest that somatic mutations are initially acquired on both strands of DNA. In this study, we utilized a previously defined trinucleotide target bias of hypermutation to address the question of target strand symmetry during mutation. Mutabilities of specific base positions within all triplets were compared between the two strands of DNA in three divergent databases of hypermutated sequences. Unexpectedly, we consistently observed strong correlations between mutabilities of triplet positions on the two DNA strands only for G and T in the first position of a triplet or for C and A in the last position. The most straightforward interpretation of this result is that the mutation mechanism targets either G and T or C and A on both strands of DNA with a frequency that depends upon the adjacent dinucleotide sequence. In view of published evidence that C is targeted by the hypermutation mechanism, we can extrapolate that C and A are specifically targeted at a frequency that depends upon the preceding 5' dinucleotide.

Extensive Deep Venous Thrombosis Resulting from Anterior Lumbar Spine Surgery in a Patient with Iliac Vein Compression Syndrome: A Case Report and Literature Review.

Study Design Case report. Objective Although May-Thurner syndrome or iliac vein compression syndrome is covered in the vascular literature, it remains absent from the orthopedic and neurosurgery literature and has not been previously reported to occur in concordance with spine surgery. We review the salient points of disease presentation, diagnosis, and treatment. Methods A 33-year-old woman was followed postoperatively via clinical and radiographic findings. Her presentation, operative treatment, postoperative extensive deep venous thrombosis (DVT) formation, and management are described. Results We present a unique case of a healthy 33-year-old woman who developed an extensive left iliac vein DVT after anterior lumbar spine fusion. Although she had multiple risk factors for thrombosis, the size of the thrombus was atypical. A subsequent venogram showed compression of the left common iliac vein by the right common iliac artery, consistent with May-Thurner syndrome. Conclusions May-Thurner syndrome or iliac vein compression syndrome is a rare diagnosis that is absent from the spine literature. The condition can predispose patients to extensive iliac vein DVT. The contributing anatomy and subsequent clot often require catheter-directed thrombolysis and stenting to achieve a favorable outcome.

Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.

The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, to release E2F transcription factors. However, this model remains unproven biochemically and the biologically active form(s) of Rb remains unknown. In this study, we find that Rb is exclusively mono-phosphorylated in early G1 phase by cyclin D:Cdk4/6. Mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but show preferential E2F binding patterns. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by quantum hyper-phosphorylation. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription, whereas cells undergoing differentiation utilize un-phosphorylated Rb. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6, is the only Rb isoform in early G1 phase.

Calcium pyrophosphate dihydrate crystal deposition disease (pseudogout) of lumbar spine mimicking osteomyelitis-discitis with epidural phlegmon.

Calcium pyrophosphate dihydrate crystal deposition disease (pseudogout) of the axial spine is rare. To our knowledge, there are few reports of the disease presenting with a presumed diagnosis of infection in the lumbar spine. As reported here, the diagnosis of osteomyelitis-discitis with epidural phlegmon was presumed before intervention. We present the case of a 60-year-old man with radiographic imaging and worsening clinical presentation at 2 consecutive hospitalizations. Axial magnetic resonance imaging originally showed increased signal intensity at the L5-S1 disk, which suggested an infectious rather than inflammatory process. Aspiration and biopsy at the time were nondiagnostic and showed no evidence of organisms. Two months after conservative treatment, the patient was readmitted with intractable low back pain and radiating bilateral leg pain. Repeat imaging showed increased interval signal in the L5-S1 disk, as well as enhancing soft-tissues that now extended to adjacent levels with extensive erosive changes. After surgical intervention for suspected infection, all cultures and stains for organisms were negative. Final pathology showed granulation tissue with focal inflammatory changes and calcium pyrophosphate crystal deposition. Although pseudogout is rare, physicians should add the disorder to the differential diagnosis for low back pain with radiculopathy and presumed infection.

High-grade adult isthmic L5-s1 spondylolisthesis: a report of intraoperative slip progression treated with surgical reduction and posterior instrumented fusion.

Adult isthmic spondylolisthesis most commonly occurs at the L5-S1 level of the lumbar spine. Slip progression is relatively rare in adults with this condition and slippage is typically associated with advanced degeneration of the disk below the pars defect. When symptomatic, radiculopathy is the typical complaint in adults with isthmic spondylolisthesis. When considering options for surgical treatment of adult isthmic spondylolisthesis, the surgeon must consider several different options, such as decompression, fusion, instrumentation, reduction, and type of bone graft to be used. All of these decisions must be individualized as deemed appropriate for each particular patient. This report presents a case of intraoperative slip progression of a L5-S1 adult isthmic spondylolisthesis to a high-grade slip, which was treated with complete surgical reduction and posterior instrumented fusion. This case demonstrates the potential instability of this condition in adults and has not been previously reported. The case details and images are reviewed and the intraoperative decisions, treatment options, and patient outcome are discussed.

Strong foreign promoters contribute to innate inflammatory responses induced by adenovirus transducing vectors.

E1-deleted adenovirus (FG Ad) transducing vectors are limited for use in vivo by their induction of strong innate and adaptive inflammatory responses. We have examined the contribution of the transgene cassette, particularly the foreign promoter driving transgene expression, in the induction of innate inflammation using a mouse ear model in which swelling is measured as a sensitive surrogate marker of the total innate inflammatory response. The commonly used cytomegalovirus major immediate early (CMV) promoter led to high-level swelling that was independent of transgene expression, while the Rous sarcoma virus and human ubiquitin C promoters led to intermediate levels of swelling and the Ad E1A promoter or no promoter led to equally low levels of swelling. Significant swelling was induced by a virus in which the E1A promoter directed pIX expression, supporting the possibility that activation of expression of Ad genes retained in the vector plays an important role in the inflammatory response. Taken together, our findings support the idea that strong foreign promoters likely play the limiting role in the induction of innate and adaptive immune responses that limit the duration of transgene expression after transduction by FG Ad vectors.

Recombinant adenoviral vectors can induce expression of p73 via the E4-orf6/7 protein.

Despite the utility of recombinant adenoviral vectors in basic research, their therapeutic promise remains unfulfilled. Most engineered adenoviral vectors use a heterologous promoter to transcribe a foreign gene. We show that adenoviruses containing the cytomegalovirus immediate-early promoter induce the expression of the proapoptotic cellular protein TAp73 via the cyclin-dependent kinase-retinoblastoma protein-E2F pathway in murine embryonic fibroblasts. Cells transduced with these vectors also expressed high levels of the adenoviral E4-orf6/7 and E2A proteins. By contrast, adenoviruses containing the ubiquitin C promoter failed to elicit these effects. E4-orf6/7 is necessary and sufficient for increased TAp73 expression, as shown by using retrovirus-mediated E4-orf6/7 expression and adenovirus with the E4-orf6/7 gene deleted. Activation of TAp73 likely occurs via E4-orf6/7-induced dimerization of E2F and subsequent binding to the inverted E2F-responsive elements within the TAp73 promoter. In addition, adenoviral vectors containing the cytomegalovirus immediate-early promoter, but not the ubiquitin C promoter, cooperated with chemotherapeutic agents to decrease cellularity in vitro. In contrast to murine embryonic fibroblasts, adenoviruses containing the ubiquitin C promoter, but not the cytomegalovirus immediate-early promoter, induced both E4-orf6/7 and TAp73 in human foreskin fibroblasts, emphasizing the importance of cellular context for promoter-dependent effects. Because TAp73 is important for the efficacy of chemotherapy, adenoviruses that increase TAp73 expression may enhance cancer therapies by promoting apoptosis. However, such adenoviruses may impair the long-term survival of transduced cells during gene replacement therapies. Our findings reveal previously unknown effects of foreign promoters in recombinant adenoviral vectors and suggest means to improve the utility of engineered adenoviruses by better controlling their impact on viral and cellular gene expression.

Multilevel spinal growth modulation with an anterolateral flexible tether in an immature bovine model.

STUDY DESIGN: A bovine model was used to evaluate the effects of a multilevel anterolateral flexible tether in a growing spine. OBJECTIVE: To evaluate the radiographic changes in a growing spine with a multilevel anterolateral tether. SUMMARY OF BACKGROUND DATA: Spinal growth modulation has long been considered as a conceptually attractive and elegant possible alternative to arthrodesis in the treatment of idiopathic scoliosis. Although some experimental studies have described spinal growth modulation, few have described a purely mechanical tether. Clinical studies of spinal epiphysiodesis have described inconsistent curve stabilization and/or correction. METHODS: A total of 33 one-month-old male calves underwent a single thoracotomy and placement of vertebral screws at T6-T9. In 11 animals, one screw per level was connected by a 3/16 in. stainless steel cable (single tether). In 11 animals, two screws per level were connected by two cables (double tether). In the remaining 11 animals, single screws in each level were left unconnected (control). After 6 months, the spines were harvested and underwent radiographic analysis. RESULTS: In the control group, there was little change in the coronal and sagittal measurements during the survival period. In the single tether group, there was variable instrumentation fixation and inconsistent creation of coronal deformity, which ranged from 0 degrees to 31 degrees. The double-tether group had more consistent creation of deformity, ranging from 23 degrees to 57 degrees. CONCLUSIONS: Given adequate bony fixation, a flexible lateral spinal tether can affect growth modulation. This technique of growth modulation may serve as a future fusionless method of correction in a growing patient with scoliosis.

Thoracic vertebral screw impingement on the aorta in an in vivo bovine model.

STUDY DESIGN: A bovine model was used to evaluate the effects of thoracic vertebral screw impingement of the aorta. OBJECTIVES: To evaluate the histologic and biomechanical changes in aortic wall tissue that was severely impinged by abutting instrumentation. SUMMARY OF BACKGROUND DATA: Case reports of vascular injury associated with spinal instrumentation generally describe intraoperative injury; some report delayed presentation of large vessel damage. Risks associated with placing instrumentation adjacent to large vessels are largely unknown. METHODS: Six 1-month-old calves underwent left-sided thoracotomies, exposing the anterior thoracic spine and aorta. With the heads removed, screws were inserted in reverse fashion into T6 through T11, leaving the screw tips 1 cm proud and abutting the aorta. After 3, 6, or 12 months (2 calves each), the spines were resected with the adjacent aorta and underwent radiographic, histologic, and biomechanical testing. RESULTS: Computed tomography revealed varying degrees of vessel impingement. Although there were no frank ruptures, 96% of aortic specimens showed histopathologic changes, including 52% with wall thinning; 43% were no longer impinged, yet 60% of these had increased collagen (scar). Biomechanical testing of screw-impinged aortas demonstrated a lower failure stress (1.2 +/- 0.5 N/mm vs. 1.8 +/- 0.4 N/mm, P = 0.016) but no difference in failure strain (42 +/- 9% vs. 32 +/- 10%, P = 0.06) than controls. CONCLUSIONS: Major impingement of vertebral screws on the aorta caused acute and chronic histopathologic and biomechanical changes in the vessel wall. This model represents a severe form of vessel penetration by a screw that confirms such a "worst case" scenario results in marked compromise of the vessel wall integrity. The sequelae of less severe impingement are unknown.

Results of surgical treatment of adult idiopathic scoliosis with low back pain and spinal stenosis: a study of long-term clinical radiographic outcomes.

STUDY DESIGN: A case series of adults with surgical treatment for adult idiopathic thoracolumbar and/or lumbar scoliosis, low back pain, and spinal stenosis was studied. OBJECTIVE: To assess pain relief, curve correction, and complications after combined procedures consisting of decompression, spine fusion, and stabilization. SUMMARY OF BACKGROUND DATA: Only one publication has focused specifically at this group, and this was before the advent of modern segmental instrumentation. This is the first report of long-term follow-up evaluation in such a patient population. METHODS: This study included 16 patients who underwent elective anterior and posterior surgical reconstruction for adult idiopathic thoracolumbar and/or lumbar scoliosis, spinal stenosis, and low back pain with a minimum follow-up period of 2 years. Radiographic findings, clinical results, and long-term outcome data were obtained using the Modified Scoliosis Research Society outcome instrument and the Oswestry Disability Back Pain Questionnaire. RESULTS: Restoration of coronal and sagittal balance, or improvement thereof, was achieved in all the patients with balance problems. There was significant improvement in all outcome domains. Overall, 94% of the patients were satisfied with the surgery. Ten major complications occurred in 10 patients, 8 of whom required additional surgery. There were two minor complications. CONCLUSIONS: Combined symptoms of back pain and spinal stenosis require complex reconstructive surgery in adults with idiopathic thoracolumbar and/or lumbar scoliosis. Significant pain relief, functional restoration, and satisfaction can be achieved and maintained over the long term in the properly selected patient.

The use of Epoetin alfa in complex spine deformity surgery.

STUDY DESIGN: A prospective, randomized trial comparing Epoetin alfa (Procrit) with placebo saline injection to determine effectiveness in increasing erythropoietic recovery in complex spine deformity surgery. OBJECTIVES: To determine if Epoetin alfa can allow preoperative autologous donation completion more effectively and reduce perioperative homologous blood transfusion. SUMMARY OF BACKGROUND DATA: The use of Epoetin alfa has been studied, primarily in the arthroplasty literature, for its effectiveness in decreasing transfusion requirements and increasing hemoglobin levels. It has not been studied in patients undergoing complex spine deformity surgery. METHODS: A total of 48 patients were prospectively randomized into an Epoetin alfa group and a control group. All patients attempted to donate 4 units of preoperative autologous donation at weekly intervals; 40,000 units of Epoetin alfa were injected subcutaneously at the time of preoperative autologous donation in the Epoetin alfa group. Hematocrit levels were recorded weekly during the donation process and daily in the preoperative period. RESULTS: Preoperative autologous donation was completed more effectively in the patients receiving Epoetin alfa. Epoetin alfa resulted in statistically higher hematocrit levels during preoperative autologous donation and perioperatively (P < 0.005). Homologous transfusion was decreased by 2.4 units and hospital stay was 1.8 days shorter in patients receiving Epoetin alfa. CONCLUSION: Patients who received Epoetin alfa were able to complete preoperative autologous donation more effectively, increase erythropoietic recovery, decrease homologous transfusion requirements, and had shorter hospital stays.

Evolution of Ig DNA sequence to target specific base positions within codons for somatic hypermutation.

Ig variable (V) region genes are subjected to a somatic hypermutation process as B lymphocytes participate in immune reactions to protein Ags. Although little is known regarding the mechanism of mutagenesis, a consistent hierarchy of trinucleotide target preferences is evident. Analysis of trinucleotide regional distributions predicted and we now empirically confirm the surprising finding that the framework 2 region of kappa V region genes is highly mutable despite its importance to the structural integrity and function of the Ab molecule. Interestingly, much of this mutability appears to be focused on the third codon position where synonymous substitutions are most likely to occur. We also observed a trend for high predicted mutability for codon positions 1 and 2 in complementarity-determining regions. Consequently, amino acid replacements should occur at a higher rate in complementarity-determining regions than in framework regions due to the distribution and subsequent targeting of microsequences by the mutation mechanism. Our results reveal a subtle tier of V region gene evolution in which DNA sequence has been molded to direct mutations to specific base positions within codons in a manner that minimizes damage and maximizes the benefits of the somatic hypermutation process.

The use of a constrained acetabular component for recurrent dislocation.

The poor results of surgical treatment of chronic instability after total hip arthroplasty (THA) led to the development of a constrained acetabular component. In this study, 87 constrained THAs implanted for recurrent instability were reviewed retrospectively. Eighty-five hips were available for follow-up evaluation, with an average follow-up period of 58 months. These 85 hips were evaluated at a minimum of 3 years. Two recurrent dislocations were seen, caused by dissociation of the liner from the shell. Four acetabular components and 1 femoral component were revised. Overall, a 2.4% dislocation rate and an 8.2% revision rate were seen. The recurrent dislocation rate of 2.4% represents a significant improvement over other methods reported. Repeat dislocation was only seen in dissociation of cemented liners into well-fixed shells. We do not recommend this mode of fixation.

Cervical epidural abscess after epidural steroid injection.

STUDY DESIGN: This is a case report of a cervical epidural abscess presenting with neurologic deficits after cervical epidural steroid injection. OBJECTIVE: To describe the presentation, diagnosis, treatment, and outcome of a rare complication of cervical epidural steroid injection. SUMMARY OF BACKGROUND DATA: Cervical epidural steroid injections are a commonly used modality in the treatment of cervical spine disease. Serious complications from the procedure are rare. There is only one previously reported case of cervical epidural abscess after cervical epidural injection in the literature. MATERIALS AND METHODS: A case of cervical epidural abscess after epidural steroid injection is presented and the relevant literature is reviewed. RESULTS: The patient had partial recovery of neurologic function within the first 24 hours after decompressive laminectomy, irrigation, and debridement. There were no perioperative complications. Intraoperative cultures permitted positive identification of the infecting organism and appropriate antibiotic selection. At 7-month follow-up, there was no recurrence of infection and the patient had recovered baseline neurologic function and neck pain status. CONCLUSIONS: Cervical epidural abscess is a rare but potentially devastating complication after epidural steroid injection. Neurologic compromise may occur. Timely diagnosis and appropriate treatment may result in good clinical outcomes.