Citations

Abstract Background: Glycerol kinase deficiency (GKD) is an X-linked recessive disorder that presents in both isolated and complex forms. The contiguous deletion that leads to GKD also commonly affects NR0B1 (DAX1), the gene associated with adrenal hypoplasia congenita, and DMD, the Duchenne muscular dystrophy gene. Molecular testing to delineate this deletion is expensive and has only limited availability. Methods: We designed a multiplex PCR assay for the detection and mapping of a contiguous deletion potentially affecting the IL1RAPL1, NR0B1, GK, and DMD genes in a 29-month-old male patient with GKD. Results: Multiplex PCR detected a contiguous deletion that involved the IL1RAPL1, NR0B1, GK, and DMD genes. Although the patient had a creatine kinase concentration within the reference interval, further mapping with PCR revealed that exon 74 was the last intact exon at the 3′ end of the DMD gene. Conclusions: Multiplex PCR is an effective and inexpensive way to detect and map the contiguous deletion in cases of complex GKD. The extension of a deletion to include DMD exon 75 in a patient with a creatine kinase concentration within the reference interval suggests that this region of the gene may not be essential for protein function.

Abstract Cancer cells secrete matrix metalloproteinases (MMP) that degrade the extracellular matrix and are responsible for some hallmarks of malignant cancer. Many viruses, including a few currently used in oncolytic virotherapy clinical trials, depend on intracellular proteases to process their proteins and activate their particles. We show here for measles virus (MV) that particle activation can be made dependent of proteases secreted by cancer cells. The MV depends on the intracellular protease furin to process and activate its envelope fusion (F) protein. To make F protein activation cancer cell specific, we introduced hexameric sequences recognized by an MMP and identified the mutant proteins most effective in fusing MMP-expressing human fibrosarcoma cells (HT1080). We showed that an MMP inhibitor interferes with syncytia formation elicited by mutant F proteins and confirmed MMP-dependent cleavage by Edman degradation sequence analysis. We generated recombinant MVs expressing the modified F proteins in place of furin-activated F. These viruses spread only in cells secreting MMP. In nude mice, an MMP-activated MV retarded HT1080 xenograft growth as efficiently as the furin-activated MV vaccine strain. In MV-susceptible mice, the furin-activated virus caused lethal encephalitis upon intracerebral inoculation, whereas the MMP-activated did not. Thus, MV particle activation can be made dependent of proteases secreted by cancer cells, enhancing safety. This study opens the perspective of combining targeting at the particle activation, receptor recognition, and selective replication levels to improve the therapeutic index of MV and other viruses in ongoing clinical trials of oncolysis. (Cancer Res 2006; 66(15): 7694-700)

Abstract MLH3 is a recently described member of the DNA mismatch repair gene family. Based on its interaction with the MutL homologue MLH1, it was postulated that MLH3 might play a role in tumorigenesis. Germ line and somatic mutations in MLH3 have been identified in a small fraction of colorectal cancers, but the role of MLH3 in colorectal cancer tumorigenesis remains controversial. We investigated MLH3's role in endometrial tumorigenesis through analysis of tumor and germ line DNA from 57 endometrial cancer patients who were at increased risk for having inherited cancer susceptibility. Patients with known MSH2 or MSH6 mutations were excluded as well as those who had MLH1-methylated tumors. Sixteen different variants were identified by single-strand conformational variant analysis. Of the 12 missense changes identified, three were somatic mutations. One patient had a germ line missense variant and loss of heterozygosity (LOH) in her tumor specimen. There was no evidence of MLH3 promoter methylation based on combined bisulfite restriction analysis. The identification of inherited missense variants, somatic missense mutations (present in 3 of 57 tumors), and LOH in the tumor from a patient with a germ line missense change suggest a role for MLH3 in endometrial tumorigenesis. (Cancer Res 2006; 66(15): 7502-8)

We describe a strategy to “revive” putatively pathogenic T cells from frozen specimens of human inflammatory target organs. To distinguish pathogenic from irrelevant bystander T cells, we focused on cells that were ( i ) clonally expanded and ( ii ) in direct morphological contact with a target cell. Using CDR3 spectratyping, we identified clonally expanded T cell receptor (TCR) β-chains in muscle sections of patients with inflammatory muscle diseases. By immunohistochemistry, we identified those Vβ-positive T cells that fulfilled the morphological criteria of myocytotoxicity and isolated them by laser microdissection. Next, we identified coexpressed pairs of TCR α- and β-chains by a multiplex PCR protocol, which allows the concomitant amplification of both chains from single cells. This concomitant amplification had not been achieved previously in histological sections, mainly because of the paucity of available anti-α-chain antibodies and the great heterogeneity of the α-chain genes. From muscle tissue of a patient with polymyositis, we isolated 64 T cells that expressed an expanded Vβ1 chain. In 23 of these cells, we identified the corresponding α-chain. Twenty of these 23 α-chains were identical, suggesting antigen-driven selection. After functional reconstitution of the αβ-pairs, their antigen-recognition properties could be studied. Our results open avenues for combined analysis of the full TCR α- and β-chain repertoire in human inflammatory tissues.

Because physician turnover, especially during the first 5 years after joining a practice group, has been reported to be as high as 54%, we sought to identify and quantify the factors that lead hand surgeons to leave their practices. An eight-question practice survey was administered to 10 hand surgeons, all of whom had left their practices under various circumstances. All respondents were men (mean age, 43.2 years). Nine surgeons were in their first practice and one was in his second. Time in the practice ranged from 4 months to 6.5 years (mean time, 3.4 years). Questions about surgical practice issues were asked of all survey participants, who were then asked to quantify the influence of the issue on their decision to leave their own surgical practice with a Likert scale ranging from 1 (no influence) to 5 (greatly influenced the decision to leave). The strongest influences on the decision to leave a practice were interpersonal conflict (mean, 4.7 points) and lack of decision-making ability within the practice (mean, 4.2 points). The influence of partnership determination (mean, 3.3 points) was mixed, whereas staff support (mean, 2.9 points), salary agreements (mean, 2.4 points), employment contracts (mean, 1.6 points), and retirement benefits (mean, 1.0 points) played a lesser role in the surgeons' decisions to leave their practices.

SummaryOne of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme-linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample-specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen-presenting cell platform for flow cytometry-based quantification of the frequency and activation status of peptide-specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)-A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine-producing) and inactive tetramer-positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high-throughput applications and automation. The utility and application of this method are discussed.