William A. LaFramboise

Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle.

One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis. Electrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to be: 2A less than 2X less than 2B less than beta/slow.

Emergence of the mature myosin phenotype in the rat diaphragm muscle

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.

MicroRNA-137 promoter methylation is associated with poorer overall survival in patients with squamous cell carcinoma of the head and neck.

The overall 5‐year survival rate of approximately 60% for head and neck cancer patients has remained essentially unchanged over the past 30 years. MicroRNA‐137 (miR‐137) plays an essential role in cell‐cycle control at the G1/S‐phase checkpoint. However, the aberrant miR‐137 promoter methylation observed in squamous cell carcinoma of the head and neck (SCCHN) suggests a tumor‐specific molecular defect that may contribute to disease progression.

Identification of differentially expressed genes in fibroblasts derived from patients with Dupuytren's Contracture

Dupuytrens contracture (DC) is the most common inherited connective tissue disease of humans and is hypothesized to be associated with aberrant wound healing of the palmar fascia. Fibroblasts and myofibroblasts are believed to play an important role in the genesis of DC and the fibroproliferation and contraction that are hallmarks of this disease. This study compares the gene expression profiles of fibroblasts isolated from DC patients and controls in an attempt to identify key genes whose regulation might be significantly altered in fibroblasts found within the palmar fascia of Dupuytrens patients. Total RNA isolated from diseased palmar fascia (DC) and normal palmar fascia (obtained during carpal tunnel release; 6 samples per group) was subjected to quantitative analyses using two different microarray platforms (GE Code Link™ and Illumina™) to identify and validate differentially expressed genes. The data obtained was analyzed using The Significance Analysis of Microarrays (SAM) software through which we identified 69 and 40 differentially regulated gene transcripts using the CodeLink™ and Illumina™ platforms, respectively. The CodeLink™ platform identified 18 upregulated and 51 downregulated genes. Using the Illumina™ platform, 40 genes were identified as downregulated, eleven of which were identified by both platforms. Quantitative RT-PCR confirmed the downregulation of three high-interest candidate genes which are all components of the extracellular matrix: proteoglycan 4 (PRG4), fibulin-1 (FBLN-1) transcript variant D, and type XV collagen alpha 1 chain. Overall, our study has identified a variety of candidate genes that may be involved in the pathophysiology of Dupuytrens contracture and may ultimately serve as attractive molecular targets for alternative therapies.

Clinical Integration of Next-Generation Sequencing Technology

Recent advances in next-generation sequencing (NGS) methods and technology have substantially reduced costs and operational complexity leading to production of benchtop sequencers and commercial software solutions for implementation in small research and clinical laboratories. This article addresses requirements and limitations to successful implementation of these systems, including (1) calibration and validation of the instrumentation, experimental paradigm, and primary readout, (2) secure data transfer, storage, and secondary processing, (3) implementation of software tools for targeted analysis, and (4) training of research and clinical personnel to evaluate data fidelity and interpret the molecular significance of the genomic output.

Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity

While Chlamydia trachomatis infections are frequently asymptomatic, mechanisms that regulate host response to this intracellular Gram-negative bacterium remain undefined. This investigation thus used peripheral blood mononuclear cells and endometrial tissue from women with or without Chlamydia genital tract infection to better define this response. Initial genome-wide microarray analysis revealed highly elevated expression of matrix metalloproteinase 10 and other molecules characteristic of Type 2 immunity (e.g., fibrosis and wound repair) in Chlamydia-infected tissue. This result was corroborated in flow cytometry and immunohistochemistry studies that showed extant upper genital tract Chlamydia infection was associated with increased co-expression of CD200 receptor and CD206 (markers of alternative macrophage activation) by endometrial macrophages as well as increased expression of GATA-3 (the transcription factor regulating TH2 differentiation) by endometrial CD4+ T cells. Also among women with genital tract Chlamydia infection, peripheral CD3+ CD4+ and CD3+ CD4- cells that proliferated in response to ex vivo stimulation with inactivated chlamydial antigen secreted significantly more interleukin (IL)-4 than tumor necrosis factor, interferon-γ, or IL-17; findings that repeated in T cells isolated from these same women 1 and 4 months after infection had been eradicated. Our results thus newly reveal that genital infection by an obligate intracellular bacterium induces polarization towards Type 2 immunity, including Chlamydia-specific TH2 development. Based on these findings, we now speculate that Type 2 immunity was selected by evolution as the host response to C. trachomatis in the human female genital tract to control infection and minimize immunopathological damage to vital reproductive structures.

A four-marker signature of TNF-RII, TGF-α, TIMP-1 and CRP is prognostic of worse survival in high-risk surgically resected melanoma

BackgroundE1694 tested GM2-KLH-QS21 vaccine versus high-dose interferon-α2b (HDI) as adjuvant therapy for operable stage IIB-III melanoma. We tested banked serum specimens from patients in the vaccine arm of E1694 for prognostic biomarkers.MethodsAushon Multiplex Platform was used to quantitate baseline serum levels of 115 analytes from 40 patients. Least absolute shrinkage and selection operator proportional hazard regression (Lasso PH) was used to select markers that are most informative for relapse-free survival (RFS) and overall survival (OS). Regular Cox PH models were then fit with the markers selected by the Lasso PH. Survival receiver operating characteristic (ROC) analysis was used to evaluate the ability of the models to predict 1-year RFS and 5-year OS.ResultsFour markers that include Tumor Necrosis Factor alpha Receptor II (TNF-RII), Transforming Growth Factor alpha (TGF-α), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), and C-reactive protein (CRP) were found to be most informative for the prediction of OS (high levels correlate with worse prognosis). The dichotomized risk score based on the four markers could significantly separate the OS curves (p = 0.0005). When using the four-marker PH model to predict 5-year OS, we achieved an area under the curve (AUC) of 89% (cross validated AUC = 72%). High baseline TNF-RII was also significantly associated with worse RFS. The RFS with high (above median) TNF-RII was significantly lower than low TNF-RII (p = 0.01).ConclusionsThe biomarker signature consisting of TNFR-II, TGF-α, TIMP-1 and CRP is significantly prognostic of survival in patients with high-risk melanoma and warrants further investigation.

Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

BackgroundDupuytrens contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC.MethodsTo achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram.ResultsWe found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder.ConclusionsThese data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.

Genome abnormalities precede prostate cancer and predict clinical relapse.

The prediction of prostate cancer clinical outcome remains a major challenge after the diagnosis, even with improved early detection by prostate-specific antigen (PSA) monitoring. To evaluate whether copy number variation (CNV) of the genomes in prostate cancer tumor, in benign prostate tissues adjacent to the tumor (AT), and in the blood of patients with prostate cancer predicts biochemical (PSA) relapse and the kinetics of relapse, 241 samples (104 tumor, 49 matched AT, 85 matched blood, and 3 cell lines) were analyzed using Affymetrix SNP 6.0 chips. By using gene-specific CNV from tumor, the genome model correctly predicted 73% (receiver operating characteristic P = 0.003) cases for relapse and 75% (P < 0.001) cases for short PSA doubling time (PSADT, <4 months). The gene-specific CNV model from AT correctly predicted 67% (P = 0.041) cases for relapse and 77% (P = 0.015) cases for short PSADT. By using median-sized CNV from blood, the genome model correctly predicted 81% (P < 0.001) cases for relapse and 69% (P = 0.001) cases for short PSADT. By using median-sized CNV from tumor, the genome model correctly predicted 75% (P < 0.001) cases for relapse and 80% (P < 0.001) cases for short PSADT. For the first time, our analysis indicates that genomic abnormalities in either benign or malignant tissues are predictive of the clinical outcome of a malignancy.

Mismatch repair deficiency is an uncommon mechanism of alkylator resistance in pediatric malignant gliomas: A report from the children's oncology group†

Alkylating agents are commonly used in the treatment of childhood malignant gliomas. Overexpression of O6‐methylguanine‐DNA methyltransferase (MGMT) constitutes an important mechanism for resistance to such agents, and MGMT status has been associated with outcome in several recent trials. Deficiency in mismatch repair (MMR) function has been implicated in preclinical studies as an additional potential mechanism of resistance to methylating agents, such as temozolomide, independent of tumor MGMT status. However, the frequency of this abnormality as a clinical resistance mechanism in childhood malignant gliomas has not been well characterized.