Multilineage potential of homozygous stem cells derived from metaphase II oocytes

Human stem cells derived from human fertilized oocytes, fetal primordial germ cells, umbilical cord blood, and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However, the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach, employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D), we established stem cell lines homozygous for H-2-B and H-2-D, respectively. The undifferentiated cells retained a normal karyotype, expressed stage-specific embryonic antigen-1 and Oct4, and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition, these cells demonstrated the potential for in vitro differentiation into endoderm, neuronal, and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes, and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible "homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation, while decreasing the ethical concerns surrounding human embryonic stem cell research.     

Elevated amyloid beta protein (Abeta42) and late onset Alzheimer's disease are associated with single nucleotide polymorphisms in the urokinase-type plasminogen activator gene

Plasma amyloid beta protein (Abeta42) levels and late onset Alzheimer's disease (LOAD) have been linked to the same region on chromosome 10q. The PLAU gene within this region encodes urokinase-type plasminogen activator, which converts plasminogen to plasmin. Abeta aggregates induce PLAU expression thereby increasing plasmin, which degrades both aggregated and non-aggregated forms of Abeta. We evaluated single nucleotide polymorphisms (SNPs) in PLAU for association with Abeta42 and LOAD. PLAU SNP compound genotypes composed of haplotype pairs showed significant association with AD in three independent case-control series. PLAU SNP haplotypes associated significantly with plasma Abeta42 in 10 extended LOAD families. One of the SNPs analyzed was a missense C/T polymorphism in exon 6 of PLAU (PLAU_1=rs2227564), which causes a proline to leucine change (P141L). We analyzed PLAU_1 for association with AD in six case-control series and 24 extended LOAD families. The CT and TT PLAU_1 genotypes showed association (P=0.05) with an overall estimated odds ratio of 1.2 (1.0-1.5). The CT and TT genotypes of PLAU_1 were also associated with significant age-dependent elevation of plasma Abeta42 in 24 extended LOAD families (P=0.0006). In knockout mice lacking the PLAU gene, plasma--but not brain--Abeta42 as well as Abeta40 was significantly elevated, also in an age-dependent manner. The PLAU_1 associations were independent of the associations we found among plasma Abeta42, LOAD and variants in the IDE or VR22 region. These results provide strong evidence that PLAU or a nearby gene is involved in the development of LOAD. PLAU_1 is a plausible pathogenic mutation that could act by increasing Abeta42, but additional biological experiments are required to show this definitively.     

Innovations in curriculum design: A multi-disciplinary approach to teaching statistics to undergraduate medical students

Background  

Statistics is relevant to students and practitioners in medicine and health sciences and is increasingly taught as part of the medical curriculum. However, it is common for students to dislike and under-perform in statistics. We sought to address these issues by redesigning the way that statistics is taught.     

Analysis of postarousal EEG activity during somnambulistic episodes

Early studies found that electroencephalographic (EEG) recordings during somnambulistic episodes were characterized by a combination of alpha, theta, and delta frequencies, without evidence of clear wakefulness. Three postarousal EEG patterns associated with slow-wave sleep (SWS) arousals were recently identified in adults with sleepwalking and sleep terrors. The goal of the present study was to evaluate the distribution of these postarousal EEG patterns in 10 somnambulistic patients (three males, seven females, mean age: 25.1, SD: 4.1) evaluated at baseline and following 38 h of sleep deprivation. A total of 44 behavioral arousals were recorded in the laboratory; seven episodes at baseline (five from SWS, two from stage 2 sleep) and 37 episodes during recovery sleep (30 from SWS, seven from stage 2 sleep). There was no significant difference in the distribution of postarousal EEG patterns identified during baseline and recovery sleep. One pattern, comprised of diffuse rhythmic and synchronous delta activity, was preferentially associated with relatively simple behavioral episodes but did not occur during episodes from stage 2 sleep. Overall, delta activity was detected in 48% of the behavioral episodes from SWS and in 22% of those from stage 2. There was no evidence of complete awakening during any of the episodes. The results support the view of somnambulism as a disorder of arousal and suggest that sleepwalkers' atypical arousal reactions can manifest themselves in stage 2 sleep in addition to SWS.     

Chromosome analyses of 16 cases of Wilms tumor: different pattern in unfavorable histology

Cytogenetic analyses of 16 cases of Wilms tumor with abnormal karyotypes were reviewed, 15 cases of unilateral tumor and 1 bilateral. Three tumors exhibited an unfavorable histology (i.e., anaplastic changes); the rest fell into the favorable histology group. Of the 17 tumors with abnormal clonal aberrations, 9 tumors were hyperdiploid (53%), 7 had pseudodiploid karyotypes (41%), and 1 was hypodiploid (6%). The most common numerical aberrations in descending order of frequency were gain of chromosomes 12, 8, and 6 and loss of chromosome 16. Structural rearrangements mostly involved chromosome 1, followed by chromosomes 7, 14, and 17. Clustering of breaks around 1p22 approximately p31-->pter resulting in partial loss of 1p was the most frequent structural aberration. Additionally, i(7q) was observed as a sole abnormality in two tumors and a 7p translocation in two other tumors. Two other recurrent abnormalities were a partial deletion of 14q, seen in three tumors, and complete loss of chromosome 14 in one tumor. All three Wilms tumors with unfavorable histology had abnormalities of 17p, resulting in TP53 gene deletion. These findings provide further support for the importance of gains of chromosomes 12, 8, and 6 and loss of 1p in the development of Wilms tumor. The results also support the association of unfavorable-histology Wilms tumors with TP53 deletion. The nonrandom losses of 16/16q, 7p, and 14q may point to the importance of genomic imbalance in the pathogenetic consequences and progression of Wilms tumor.     

Anti-tumor immunoglobulin M increases lung metastasis in an experimental model of malignant melanoma

Cancer metastasis involves distinct steps that depend on complicated tumor–host interactions. The hematogenous dissemination of tumor cells may be facilitated by factors that promote the arrest and adherence of cancer cells in capillaries. We examined whether anti-tumor monoclonal immunoglobulin M (IgM) antibodies promoted the hematogenous dissemination of B16 melanoma cells in syngeneic mice. IgM monoclonal antibodies were generated that selectively bind to B16 melanoma cells as compared to syngeneic fibroblasts, lymphocytes or Lewis lung carcinoma cells. Incubation of B16-BL6 or B16-F0 melanoma cells with these IgM anti-tumor antibodies significantly increased the number of lung colonies as compared with control antibodies. Moreover, intraperitoneal injection of specific antibody also significantly increased lung colonization. All anti-tumor antibodies promoted the aggregation of B16 melanoma cells. A chemically generated immunoglobulin G (IgG)-like fragment of an anti-tumor IgM antibody displayed greatly reduced tumor aggregation and, in contrast to intact IgM, did not significantly increase lung colonization of B16 melanoma cells. Neither intact IgM nor the IgG-like fragment enhanced the in vitro invasiveness of B16 melanoma cells across Matrigel-coated membranes. Our results, therefore, suggest that besides their beneficial anti-tumor effects, anti-tumor IgM antibodies may also promote the hematogenous dissemination of cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c

Few human CD8(+) T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8(+) T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha.     

Electromyographic and affective responses of episodic tension-type headache patients and headache-free controls during stressful task performance

Thirty-four subjects meeting diagnostic criteria for episodic tension-type headache and 42 who rarely experienced headaches participated in two laboratory sessions in which cephalic electromyographic (EMG) activity, electrodermal activity, heart rate, and finger temperature were recorded. Subjects performed relaxation, choice reaction time, psychomotor tracking, voluntary muscle contraction, and cold pressor tasks. Headache subjects showed significantly greater EMG activity than controls during baseline and stressful task performance. During relaxation, both groups reduced EMG activity from baseline levels, and there was no significant difference in EMG level between the groups during relaxation. Headache subjects reported higher levels of subjective anxiety, depression, anger, and stress than controls. Headache subjects also reported higher levels of pain than controls, and headache subjects reported greater pain during stressful task performance relative to baseline and recovery periods.This research was supported by NIH Grant R01-NS-25114.