James B. Hayden

T-cell receptor variable β genes show differential expression in CD4 and CD8 T cells

Abstract Studies in transgenic and inbred strains of mice have shown that the critical molecular interactions controlling positive selection involve major histocompatibility complex (MHC), T-cell receptor (TCR), and CD4 or CD8 coreceptor molecules. Correlations have been established between MHC gene products and the percentage of CD4 or CD8 T cells that express specific variable (V) β-gene products as part of the αβ heterodimer. These studies have important implications regarding potential mechanisms of HLA-linked autoimmune diseases in humans. If similar interactions are required for positive selection in humans, one would predict that the TCR repertoire expressed by mature, peripheral blood CD4 and CD8 T cells would vary. To test this hypothesis the expression of specific TCR Vβ-region genes by CD4 and CD8 T cells from healthy individuals was compared using both triple-color flow cytometry and polymerase chain reaction based experimental approaches. The results show that the TCR repertoire does vary as a function of CD4 and CD8 T-cell subsets. Among unrelated individuals certain Vβ genes were consistently overrepresented in the CD4 population (Vβ-5.1, −6.7a, and −18); some were skewed to the CD8 population (Vβ-14) while others showed variable patterns (Vβ-12 and −17). Deletion of entire Vβ gene families was not observed suggesting that this is a rare event in humans. Attempts to correlate the expressed TCR repertoire in humans with HLA alleles will require consideration of these differences in expression as a function of subset.

Phase I Trial of Preoperative Chemoradiation Plus Sorafenib for High Risk Extremity Soft Tissue Sarcomas with Dynamic Contrast-Enhanced MRI Correlates

Purpose: We conducted a phase I trial of the addition of sorafenib to a chemoradiotherapy regimen in patients with high-risk (intermediate/high grade, >5 cm) extremity soft tissue sarcoma undergoing limb salvage surgery. We conducted a correlative study of quantitative dynamic contrast-enhanced MRI (DCE-MRI) to assess response to treatment. Experimental Design: Patients were treated at increasing dose levels of sorafenib (200 mg daily, 400 mg daily, 400 mg twice daily) initiated 14 days before three preoperative and three postoperative cycles of epirubicin/ifosfamide. Radiation (28 Gy) was administered during cycle 2 with epirubicin omitted. The primary objective was to determine the maximum tolerated dose (MTD) of sorafenib. DCE-MRI was conducted at baseline, after 2 weeks of sorafenib, and before surgery. The imaging data were subjected to quantitative pharmacokinetic analyses. Results: Eighteen subjects were enrolled, of which 16 were evaluable. The MTD of sorafenib was 400 mg daily. Common grade 3–4 adverse events included neutropenia (94%), hypophosphatemia (75%), anemia (69%), thrombocytopenia (50%), and neutropenic fever/infection (50%). Of note, 38% developed wound complications requiring surgical intervention. The rate of ≥95% histopathologic tumor necrosis was 44%. Changes in DCE-MRI biomarker ΔKtrans after 2 weeks of sorafenib correlated with histologic response (R2 = 0.67, P = 0.012) at surgery. Conclusion: The addition of sorafenib to preoperative chemoradiotherapy is feasible and warrants further investigation in a larger trial. DCE-MRI detected changes in tumor perfusion after 2 weeks of sorafenib and may be a minimally invasive tool for rapid assessment of drug effect in soft tissue sarcoma. Clin Cancer Res; 19(24); 6902–11. ©2013 AACR.

Histologic response of dose-intense chemotherapy with preoperative hypofractionated radiotherapy for patients with high-risk soft tissue sarcomas

The authors studied a dose‐intense regimen of epirubicin and ifosfamide with hypofractionated preoperative radiotherapy for high‐risk soft tissue sarcomas. The primary objective was estimation of the rate of ≥95% pathologic necrosis.

ANALYSIS OF NATURALLY PROCESSED PEPTIDES ELUTED FROM HLA DRB1*0402 AND *0404

Understanding the structural features of naturally processed peptides found within the major histocompatibility complex (MHC) class II peptide binding groove from disease‐associated MHC molecules may provide insights into the nature of potential disease‐related antigens. Class II MHC/peptide complexes were purified by immunoaffinity from transformed B cell lines homozygous for DRB1*0404 (an allele associated with rheumatoid arthritis) and *0402 (a closely related allele not associated with this disease). Peptides were eluted at acidic pH, fractionated by reversed phase HPLC, and analyzed by capillary electrophoresis. Those fractions containing a single dominant peptide were sequenced by automated Edman degradation and tandem mass spectrometry. The predominant peptide species identified came from non‐polymorphic regions of the HLA class I molecules expressed by each cell line. Peptides from DRB1*0404 were found to be nested clusters derived from positions 26–43 of the HLA‐B and ‐C α‐chain. DRB1*0402 contained as the predominant peptide species a nested cluster from positions 129–145 of the HLA‐B α‐chain. The primary structure of the class I derived peptides was consistent with that seen by peptides exhibiting promiscuous DR binding behavior. Processing of MHC‐derived peptides by MHC class II molecules is a common occurrence in the transformed B cell lines analyzed.

Multiple Cultures and Extended Incubation for Hip and Knee Arthroplasty Revision: Impact on Clinical Care

The impact on patient care of introducing a protocol of obtaining 5 or more intra-operative separate tissue biopsies that were cultured for 10 days was assessed for hip and knee arthroplasty revision. The charts of seventy-three patients undergoing 77 cases of revision arthroplasty were reviewed one year post-operatively. When compared to the prior standard of obtaining only one intra-operative culture, the protocol changed the microbiological diagnosis in 26/77 cases (34%, 95% Confidence Interval (CI): 23-45%) and antibiotic treatment in 23/77 cases (30%, 95% CI: 20-41%). In addition, the protocol had a predictive value of joint sterility in culture negative cases of 95% (95% CI: 85-99%). This data demonstrated the new protocol significantly changed patient care, and suggests that 1 or 2 cultures are insufficient. Adopting a similar protocol should be considered by surgeons and institutions as a new minimum standard for management of prosthetic joint infections.

Sequencing Peptides Derived from the Class II Major Histocompatibility Complex by Tandem Mass Spectrometry

Publisher Summary This chapter discusses the sequencing peptides derived from the class II major histocompatibility complex by tandem mass spectrometry (MS). Micro column reverse-phase HPLC electrospray ionization tandem MS is a rapid and sensitive technique for the analysis of complex mixtures of peptides. This technique is used to determine the amino acid sequence of unknown peptides, to verify the structure of proteins, and to determine posttranslational modifications. Under low-energy, multiple collision conditions, peptides fragment primarily at the amide bonds, producing sequence-specific fragmentation patterns. Subtraction of the mass-to-charge ratios (m/z) for consecutive type b- or y-sequence ions gives a residue mass that corresponds to the amino acid in the larger fragment. Thus, fragment ions have a cluster appearance created by selection of the entire isotopes for the precursor ion and, in part, by the computer algorithm that defines peaks and assigns masses.

What Factors Are Associated With Failure of Compressive Osseointegration Fixation

BackgroundCompressive osseointegration is as an alternative to traditional intramedullary fixation. Two- to 10-year survivorship and modes of failure have been reported; however, as a result of relatively small numbers, these studies are limited in their ability to identify risk factors for failure.Questions/purposes(1) What is survivorship free from aseptic mechanical and survivorship free from overall failure of compressive osseointegration fixation? (2) What patient factors (age, sex, body mass index [BMI], anatomic location of reconstruction, indication for reconstruction, radiation, chemotherapy) are associated with increased risk of failure?MethodsBetween 2006 and 2014, surgeons at one center treated 116 patients with 137 Compress® implants for lower extremity oncologic reconstructions, revision arthroplasty, and fracture nonunion or malunion. One hundred sixteen implants were available for review with a minimum of 2-year followup (mean, 4 years; range, 2–9 years). Kaplan-Meier survival plots were produced to examine survivorship and Cox regression modeling was used to generate hazard ratios (HRs) for potential risk factors for failure. Patient factors (age, sex, BMI, anatomic location of reconstruction, indication for reconstruction, radiation, chemotherapy) were obtained from chart review and an institutional database.ResultsSurvivorship free from aseptic mechanical failure was 95% (95% confidence interval [CI], 91%–99%) at 18 months and 93% (95% CI, 86%–99%) at 4 years. Survivorship free from overall failure was 82% (95% CI, 75%–89%) at 18 months and 75% (95% CI, 66%–84%) at 4 years. Risk of overall failure was increased with reconstruction of the proximal tibia (HR, 4.42; 95% CI 0.98–19.9) and distal femur (HR, 1.74; 95% CI, 0.50–6.09) compared to the proximal femur (HR, 1; referent; p = 0.049). Risk of aseptic mechanical failure was increased with reconstruction of the proximal tibia (HR, 1; referent) and distal femur (HR, 0.37; 95% CI, 0.08–1.77) compared with the proximal femur (HR, 0, p = 0.048). Radiation was associated with increased risk of overall failure (HR, 3.85; 95% CI, 1.84–8.02; p < 0.003), but not aseptic mechanical failure. Age, sex, BMI, chemotherapy, and surgical indication were not associated with increased risk of aseptic or overall failure.ConclusionsThis study questions the use of age as a contraindication for the use of this technology and suggests this technology may be considered in proximal femoral reconstruction and for patients with indications other than primary oncologic reconstructions. Future research should establish long-term survivorship data to compare this approach with conventional intramedullary stems and to evaluate the potential benefits of preventing stress shielding and preserving bone stock in revision situations.Level of EvidenceLevel III, therapeutic study.

Preclinical testing of the glycogen synthase kinase-3β inhibitor tideglusib for rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3β is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3β inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models in vivo. In this study, we tested the irreversible GSK3β-inhibitor, tideglusib for in vivo efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3β inhibitors may not be a viable treatment for aRMS or eRMS.Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. RMS often arise from myogenic precursors and displays a poorly differentiated skeletal muscle phenotype most closely resembling regenerating muscle. GSK3β is a ubiquitously expressed serine-threonine kinase capable of repressing the terminal myogenic differentiation program in cardiac and skeletal muscle. Recent unbiased chemical screening efforts have prioritized GSK3β inhibitors as inducers of myodifferentiation in RMS, suggesting efficacy as single agents in suppressing growth and promoting self-renewal in zebrafish transgenic embryonal RMS (eRMS) models in vivo. In this study, we tested the irreversible GSK3β-inhibitor, tideglusib for in vivo efficacy in patient-derived xenograft models of both alveolar rhabdomyosarcoma (aRMS) and eRMS. Tideglusib had effective on-target pharmacodynamic efficacy, but as a single agent had no effect on tumor progression or myodifferentiation. These results suggest that as monotherapy, GSK3β inhibitors may not be a viable treatment for aRMS or eRMS.