Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection

We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8(pos) and also CD4(pos) cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection.     

The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans.

The lin-4 gene encodes a small RNA that is required to translationally repress lin-14 toward the end of the first larval stage of Caenorhabditis elegans development. To determine if the timing of LIN-14 protein down-regulation depends on the temporal profile of lin-4 RNA level, we analyzed the stage-specificity of lin-4 RNA expression during wild-type development and examined the phenotypes of transgenic worms that overexpress lin-4 RNA during the first larval stage. We found that lin-4 RNA first becomes detectable at approximately 12 h of wild-type larval development and rapidly accumulates to nearly maximum levels by 16 h. This profile of lin-4 RNA accumulation corresponded to the timing of LIN-14 protein down-regulation. Transgenic strains that express elevated levels of lin-4 RNA prior to 12 h of development display reduced levels of LIN-14 protein and precocious phenotypes consistent with abnormally early loss of lin-14 activity. These results indicate that the temporal profile of lin-4 RNA accumulation specifies the timing of LIN-14 down-regulation and thereby controls the timing of postembryonic developmental events.     

Chronopharmacological Study of an Antimicrobial Agent,Norfloxacin, in the Rat

Circadian rhythms in physiological processes may affect pharmacological actions of drugs. The purpose of this study was to determine whether pharmacokinetics or acute lethality (LD 50) of norfloxacin, exhibited circadian rhythmicity. Female Sprague- Dawley prepuberal rats (weight 115.8 ± 10.2 g) synchronized with a 12-h-light/ 12-h-dark cycle (lights on 7:00h) were used throughout the study. Norfloxacin pharmacokinetics after intraperitoneal administration at 4:00, 10:00, 16:00 and 22:00h was characterized. Intraperitoneal norfloxacin LD 50 was administered at 2:00, 6:00, 10:00, 14:00, 18:00 and 22:00 h. Pharmacokinetic parameters and lethality percentages were analyzed by the cosinor method for the presence of circadian rhythmicity. The results showed evidence of circadian rhythmicity for norfloxacin k abs, t ½abs, t max, MRT abs, Cl t /f and AUC. Absorption was higher when the drug was administered during the rest (16:00 h) period, meanwhile elimination was higher when administered during the activity (22:00 h) period. No rhythmicity was determined for norfloxacin lethality. It is concluded that, in this study, time of administration modifies the pharmacokinetics of norfloxacin.     

MicroRNAs与疾病和发育

作为模式生物实验系统,线虫可用于研究控制动物发育和人类疾病遗传机制。研究发育缺陷的线虫突变体有助于在动物中发现对发育和生理过程有重要调控作用的基因。其中一些基因编码一类小RNA,如microRNA(miRNA),通过作用于特定基因信使RNA来调控其蛋白质表达。一些在线虫发育过程中有功能的miRNA在人体中也存在。它们参与调控与疾病相关的生物学过程,如癌症、糖尿病和神经退行性疾病。通过分析miRNA在临床样品、哺乳动物细胞和模式生物线虫中的表达,从而揭示miRNA调控途径在相关人类疾病中的功能。     

Nuclear morphometry as a prognostic indicator in colorectal carcinoma resected for cure

The prognostic value of nuclear morphometry in addition to clinical and pathologic features was retrospectively studied in 64 cases of colorectal carcinoma resected for cure with a minimum of five years of follow-up. By univariate analysis, patient outcome was found to correlate with the presence of serosal involvement (P = .003), the presence of lymph node involvement (P = .01), the number of involved lymph nodes (P = .0001) and the mean nuclear area (P = .02). With multivariate analysis, only the number of involved lymph nodes significantly correlated with the survival (P = .0001). In a subsequent multivariate model expressing lymph node status as the presence or absence of metastasis, the presence or absence of serosal involvement and the mean nuclear area were both found to independently correlate with the outcome (P = .003 and P = .02, respectively). Linear regression analysis revealed significant colinearity between the mean nuclear area and the number of involved lymph nodes (P = .03). Accelerated failure time models based on determination of serosal involvement and then either specification of the number of involved lymph nodes or calculation of the mean nuclear area were of comparable predictive value to the determination of the number of involved lymph nodes alone. The former appeared to be better at identifying a subgroup of patients with good prognosis. This study demonstrates that two or more models based on pathologic features may be of comparable predictive value in colorectal carcinoma resected for cure, including models that incorporate mean nuclear area.     

Pseudomonas aeruginosa cleaves the decoding center of Caenorhabditis elegans ribosomes

Pathogens such as Pseudomonas aeruginosa advantageously modify animal host physiology, for example, by inhibiting host protein synthesis. Translational inhibition of insects and mammalian hosts by P. aeruginosa utilizes the well-known exotoxin A effector. However, for the infection of Caenorhabditis elegans by P. aeruginosa, the precise pathways and mechanism(s) of translational inhibition are not well understood. We found that upon exposure to P. aeruginosa PA14, C. elegans undergoes a rapid loss of intact ribosomes accompanied by the accumulation of ribosomes cleaved at helix 69 (H69) of the 26S ribosomal RNA (rRNA), a key part of ribosome decoding center. H69 cleavage is elicited by certain virulent P. aeruginosa isolates in a quorum sensing (QS)–dependent manner and independently of exotoxin A–mediated translational repression. H69 cleavage is antagonized by the 3 major host defense pathways defined by the pmk-1, fshr-1, and zip-2 genes. The level of H69 cleavage increases with the bacterial exposure time, and it is predominantly localized in the worm’s intestinal tissue. Genetic and genomic analysis suggests that H69 cleavage leads to the activation of the worm’s zip-2-mediated defense response pathway, consistent with translational inhibition. Taken together, our observations suggest that P. aeruginosa deploys a virulence mechanism to induce ribosome degradation and H69 cleavage of host ribosomes. In this manner, P. aeruginosa would impair host translation and block antibacterial responses.

During infection of the nematode Caenorhabditis elegans by the bacterium Pseudomonas aeruginosa, a bacterial virulence mechanism leads to the cleavage of host ribosomal RNAs at the decoding center, thereby shutting down translation.     

B and C Types Natriuretic Peptides Modify Norepinephrine Uptake and Release in the Rat Adrenal Medulla

Vatta, M. S., M. F. Presas, L. G. Bianciotti, M. Rodriguez–fermepin, R. Ambros and B. E. Fernandez. B and C types natriuretic peptides modify norepinephrine uptake and release in the rat adrenal medulla. Peptides 18(10) 1483–1489, 1997.—We have previously reported that atrial natriuretic factor (ANF) modulates adrenomedullar norepinephrine (NE) metabolism. On this basis, the aim of the present work was to study the effects of B and C types natriuretic peptides (BNP and CNP) on the uptake, intracellular distribution and release of ³H-NE. Experiments were carried out in rat adrenal medulla slices incubated “in vitro.” Results showed that 100 nM of both, CNP and BNP, enhanced total and neuronal NE uptake. Both peptides (100 nM) caused a rapid increase in NE uptake during the first minute, which was sustained for 60 min. NE intracellular distribution was only modified by CNP (100 nM), which increased the granular fraction and decreased the cytosolic pool. On the other hand, spontaneous as well as evoked (KCl) NE release, was decreased by BNP and CNP (50 and 100 nM for spontaneous release and 1, 10, 50 and 100 nM for evoked output). The present results suggest that BNP and CNP may regulate catecholamine secretion and modulate adrenomedullary biological actions mediated by catecholamines, such as blood arterial pressure, smooth muscle tone, and metabolic activities.