Ablation of a specific cell population by the replacement of a uniquely expressed gene with a toxin gene

The transgenic expression of a toxin gene or a thymidine kinase gene under the control of cell type-specific promoter/enhancer has been shown to be useful for removing a specific cell population in mice. However, this approach requires extensive analysis of the control elements for gene expression in the preparation of the transgenic constructs, and furthermore, the toxin gene might be expressed ectopically because of random integration, resulting in aberrant depletion of unrelated cells. To avoid such difficulties with the transgenic approach, we established a method for the specific depletion of a cell population by replacing a uniquely expressed gene in the population with the diphtheria toxin gene by using homologous recombination. The NKR-P1 gene, a specific cell surface marker of natural killer (NK) cells, was selected as the target gene for depleting NK cells. In chimeric mice reconstituted with embryonic stem cells in which the NKR-P1 gene was replaced by the toxin gene, NKR-P1(+) cells were almost completely depleted, and NK cell function was abrogated in the embryonic stem cell-derived lymphoid cells. Other cell lineages developed normally. These results show that all NK cells express NKR-P1, that NKR-P1(+) cells do not influence the development of T and B cells, and further, that this technology of cell targeting is a fast and powerful method of generating mice lacking any chosen cell population.     

Electrophysiologic parameters to predict clinical recurrence of ventricular tachycardia in patients under electrophysiologic study-guided effective pharmacological therapy.

Although an electrophysiologic study (EPS) is the most reliable method for selecting the treatment for a patient with sustained ventricular tachycardia (VT), VT recurrence may occur even during EPS-guided effective therapy. Electrophysiologic parameters were compared between patients with and without arrhythmic events under EPS-guided effective therapy to identify the predictive parameters of VT recurrence during the clinical course. The study population consisted of 77 consecutive patients with sustained VT who were receiving long-term pharmacological therapy that was demonstrated to be effective by the EPS assessment. The VT induction protocol employed 1-3 extrastimuli and rapid ventricular pacing at 2 right ventricular sites and 1 left ventricular site, and isoproterenol was infused when VT was not induced. To determine the 'effective' antiarrhythmic drug, all sustained ventricular arrhythmias had to be prevented during the whole induction protocol, but repetitive ventricular responses (RVR) were allowed to remain for up to 5 beats when they were in the same QRS configurations as the clinical VT and up to 12 beats when they were in polymorphic QRS configurations. The effective refractory periods (ERPs) at the 3 ventricular pacing sites and their difference (i.e., ERP-dispersion) and the maximum number of RVR beats were evaluated in an EPS during the control state and at the time of drug assessment. In the comparison of patients with and without VT recurrence, there was no significant difference in clinical characteristics or ERPs, but the deltaERP-dispersion (i.e., the increase in ERP-dispersion caused by the antiarrhythmic drug) and the maximum number of RVRs were significantly smaller in the group of patients without VT recurrence (deltaERP-dis, -3+/-8 vs. 6+/-12, p = 0.0027; maxRVR, 3+/-3 vs. 5+/-4, p = 0.0160). The VT recurrence rate was significantly lower in the patients with deltaERP-dis < or =0 or maxRVR<6 in comparison with the others (p = 0.01 14 and p = 0.0360). Patients with VT recurrence showed greater deltaERP-disp and a longer duration of RVRs at the time of drug assessment in comparison with the patients without VT recurrence. The prognosis of patients under EPS-guided therapy may be improved by the use of stricter criteria for drug assessment in the EPS, although this may decrease the number of drug responders determined in the EPS.     

Angiotensin II stimulation of Ca2+-channel current in vascular smooth muscle cells is inhibited by lavendustin-A and LY-294002

 Angiotensin II (AngII) is coupled to several important intracellular signaling pathways, and increases intracellular Ca²⁺. In vascular smooth muscle (VSM) cells, AngII is known to activate enzymes such as tyrosine protein kinase (Tyr-PK), phospholipase C (PLC), protein kinase C (PKC), and phophatidylinositol-3-kinase (PI-3-K). A non-receptor Tyr-PK, pp60^c-src, and PKC have been reported to stimulate the Ca²⁺ channels in VSM cells. However, less is known about AngII action on the voltage-gated Ca²⁺ channels. The Ca²⁺-channel currents of a cultured rat aortic smooth muscle cell line, A7r5, were recorded using whole-cell voltage clamp. Application of 50 nM AngII significantly increased the amplitude of Ba²⁺ currents through the voltage-gated Ca²⁺ channels (I _Ba) by 34.5±9.1% (n=10) within 1 min. In the presence of lavendustin-A (5 μM), a selective inhibitor of Tyr-PK, AngII failed to stimulate I _Ba (n=5). AngII stimulation of I _Ba was also prevented by (5 μM) LY-294002, an inhibitor of PI-3-K (n=5). In contrast, H-7 (30 μM), an inhibitor of PKC, did not prevent the effect of AngII on I _Ba (n=6). These results suggest that AngII may stimulate the Ca²⁺ channels of VSM cells through Tyr-PK and PI-3-K under conditions that probably exclude participation of PK-C. Received: 17 July 1998 / Received after revision: 10 September 1998 / Accepted: 29 September 1998     

New gut associated lymphoid tissue “cryptopatches” breed murine intestinal intraepithelial T cell precursors

Numerous mouse intraepithelial T cells (IEL) bearing either TCR-αΒ or TCR-γδ have been shown to develop somewhere in the intestinal mucosa without passing through the thymus. However, just where these T cells develop has been much less clear and has remained an open question to date. In an effort to investigate this issue, we carried out immunohistochemical study on the murine gastrointestinal tract and identified numerous tiny lymphoid tissues (~1,650 tissues/intestine) in the cryptal region of the small and large intestinal mucosa except for the stomach in which clusters of c-kit⁺ IL-7R⁺ Thy1⁺ lympho-hemopoietic progenitors accumulated (cryptopatches). The cryptopatch cells isolated from the small intestine, which were c-kit positive (c-kit⁺) but lineage marker negative (Lin), gave rise to TCR-αΒ and TCR-γδ IELs following in vivo transfer or tissue engraftment into 2 Gy-irradiated severe combined immunodeficient mice. In contrast, cells isolated from Peyer’s patches and mesenteric lymph nodes, which belong in the same intestinal immune compartment but lack c-kit⁺Lin^- cells, failed to do so. These results in conjunction with the findings of electron microscopic analysis provide direct evidence of a local intestinal T cell precursor that develops in the cryptopatches.     

Short-term exercise training improves diaphragm antioxidant capacity and endurance

These experiments tested the hypothesis that short-term endurance exercise training would rapidly improve (within 5 days) the diaphragm oxidative/antioxidant capacity and protect the diaphragm against contraction-induced oxidative stress. To test this postulate, male Sprague-Dawley rats (6 weeks old) ran on a motorized treadmill for 5 consecutive days (40–60 min · day⁻¹) at approximately 65% maximal oxygen uptake. Costal diaphragm strips were excised from both sedentary control (CON, n=14) and trained (TR, n=13) animals 24 h after the last exercise session, for measurement of in vitro contraction properties and selected biochemical parameters of oxidative/antioxidant capacity. Training did not alter diaphragm force-frequency characteristics over a full range of submaximal and maximal stimulation frequencies (P > 0.05). In contrast, training improved diaphragm resistance to fatigue as contraction forces were better-maintained by the diaphragms of the TR animals during a submaximal 60-min fatigue protocol (P < 0.05). Following the fatigue protocol, diaphragm strips from the TR animals contained 30% lower concentrations of lipid hydroperoxides compared to CON (P < 0.05). Biochemical analysis revealed that exercise training increased diaphragm oxidative and antioxidant capacity (citrate synthase activity +18%, catalase activity +24%, total superoxide dismutase activity +20%, glutathione concentration +10%) (P < 0.05). These data indicate that short-term exercise training can rapidly elevate oxidative capacity as well as enzymatic and non-enzymatic antioxidant defenses in the diaphragm. Furthermore, this up-regulation in antioxidant defenses would be accompanied by a reduction in contraction-induced lipid peroxidation and an increased fatigue resistance. Accepted: 6 August 1999     

Extent of pathologic invasion of the inferior vena cava in resected liver cancer compared with possible caval invasion diagnosed by preoperative images

The extent of cancerous invasion of the inferior vena cava (IVC) determined from resected liver cancer was examined pathologically. Ten patients presenting with liver cancer (metastatic liver cancer, five patients; hepatocellular carcinoma, three; and cholangiocellular carcinoma, two) were diagnosed with positive IVC invasion using preoperative imaging techniques of extracorporeal ultrasonography, computed tomography, magnetic resonance imaging, and vena cavography. The diagnostic criterion for positive IVC invasion by preoperative imaging was longitudinal IVC compression measuring over 50 mm, or transverse IVC compression extending to more than half the circumference of the IVC, or the presence of lesions protruding into the IVC lumen, or the presence of developed collateral veins. All patients underwent combined resection of the IVC. However, pathology results revealed that four of the ten patients had no cancerous invasion of the IVC, and that the extent of invasion along both the longitudinal and transverse axes of the IVC was much smaller than the compression shown by imaging results. We believe that detailed preoperative assessment, using a more precise imaging technique, as well as further intraoperative examination, is required to predict the full pathological extent of cancerous invasion of the IVC.     

Predictive Factors for Pneumomediastinum During Management of Connective Tissue Disease-related Interstitial Lung Disease: A Retrospective Study

Objective To identify factors associated with pneumomediastinum during management of connective tissue disease (CTD)-related interstitial lung disease (ILD). Methods Patients diagnosed with pneumomediastinum after the initiation of corticosteroid therapy for their CTD-ILD were enrolled. The baseline characteristics of patients who developed pneumomediastinum after the initiation of corticosteroid therapy (n=13, all occurring within 120 days) were compared to those of patients who did not develop pneumomediastinum (n=49). A multivariate logistic regression analysis was performed to identify factors associated with pneumomediastinum. A receiver operating characteristic (ROC) curve analysis was also performed to assess the predictive performance. Results The body mass index (BMI) [odds ratio (OR) (95% confidence interval (CI)) 0.482 (0.272-0.853)] and serum lactate dehydrogenase (LDH) [OR (95% CI) 1.013 (1-1.025)] levels at baseline were identified as independent factors associated with pneumomediastinum after corticosteroid initiation. The optimal cut-off points of the BMI and LDH levels for predicting pneumomediastinum development, as estimated by the Youden index, were 20.2 kg/m² and 378 U/L, respectively. LDH showed a sensitivity of 61.5% and the highest specificity of 87.8%. Importantly, combining these markers resulted in the highest sensitivity of 100% and a specificity of 71.4%. Conclusion A low BMI and high serum LDH levels at baseline are useful predictive factors for pneumomediastinum development in CTD-ILD patients.     

Quantitative assessment reveals the dominance of duplicated sequences in germline-derived extrachromosomal circular DNA

Extrachromosomal circular DNA (eccDNA) originates from linear chromosomal DNA in various human tissues under physiological and disease conditions. The genomic origins of eccDNA have largely been investigated using in vitro–amplified DNA. However, in vitro amplification obscures quantitative information by skewing the total population stoichiometry. In addition, the analyses have focused on eccDNA stemming from single-copy genomic regions, leaving eccDNA from multicopy regions unexamined. To address these issues, we isolated eccDNA without in vitro amplification (naïve small circular DNA, nscDNA) and assessed the populations quantitatively by integrated genomic, molecular, and cytogenetic approaches. nscDNA of up to tens of kilobases were successfully enriched by our approach and were predominantly derived from multicopy genomic regions including segmental duplications (SDs). SDs, which account for 5% of the human genome and are hotspots for copy number variations, were significantly overrepresented in sperm nscDNA, with three times more sequencing reads derived from SDs than from the entire single-copy regions. SDs were also overrepresented in mouse sperm nscDNA, which we estimated to comprise 0.2% of nuclear DNA. Considering that eccDNA can be integrated into chromosomes, germline-derived nscDNA may be a mediator of genome diversity.

In eukaryotes, the vast majority of genetic information is encoded in the linear chromosomes of the nucleus where circular DNA elements, or extrachromosomal circular DNA (eccDNA), are also present (1). eccDNA could modulate the genetic information of linear chromosomes and, as a result, phenotypes (2–5). This is the case with the most extensively studied extrachromosomal circular chromosomes, double minutes (DMs), which are several megabases (Mb) in size and are heavily implicated in cancer progression (2). DMs carry amplified oncogenes and DNA regulatory elements such as enhancers (3, 4) that promote the therapeutic resistance and progression of tumors (5). This phenotypic plasticity is associated with the remodeling of cancer genomes, as DMs can emerge from linear chromosomes, circularize, and reintegrate back into the genome (6, 7). In contrast, the biological significance of small-sized eccDNA populations found in both cancer and noncancer genomes remains unclear despite the recent surge of nucleotide-level data from next-generation sequencing (NGS)-based studies (8–15).Uncovering the expanding biological roles of eccDNA necessitates a delineation of their characteristics, including their chromosomal compositions and prevalence in cells. A barrier to fully understanding eccDNA biology could be associated with eccDNA enrichment approaches that rely on multiple displacement amplification (MDA), a non-PCR technique that amplifies DNA with a highly processive Φ29 DNA polymerase (16). When the template DNA is circular, DNA synthesis continues indefinitely, which is known as rolling-circle amplification (RCA). While RCA amplifies circular DNA, it often overrepresents small-sized populations of eccDNA (17, 18), rendering the analysis of in vitro–amplified DNA qualitative and not quantitative. RCA also eliminates epigenetic information associated with template DNA, leaving crucial biological information unaddressed. Furthermore, calling eccDNA is based on identifying sequencing reads that span the fusion points of circular DNA, which requires that discordant reads are confidently mapped to single-copy genomic regions (19).To overcome these issues and gain a broader scope of eccDNA characteristics, we developed a strategy to enrich endogenous eccDNA populations at the native state without RCA, which we designated as naïve small circular DNA (nscDNA). The purity of nscDNA populations was rigorously validated by both internal (mitochondrial DNA) and exogenous (plasmid) controls. We integrated molecular, cytogenetic, and genomic approaches to further validate and examine the physical properties and genomic origins of nscDNA. We found that human nscDNA ranges from ∼0.5 to greater than 10 kilobases (kb), encompassing relatively unexplored size populations of circular DNA between microDNA (<400 base pairs [bp]) and DMs. We rigorously examined the genomic origins of nscDNA beyond single-copy regions and found that nscDNA is predominantly derived from duplicated sequences of the human genome including segmental duplications (SDs). SDs represent recently duplicated genomic regions and are considered to be actively evolving areas of the human genome, as they are strongly associated with copy number variations and contribute more to genetic diversity in humans than single nucleotide polymorphisms (20–22). The strong association of duplicated sequences with genetic diversity prompted us to study germline-derived nscDNA in human and mouse sperm and tissues, which we found to be overrepresented with SDs.