Inhibition on LS-174T cell growth and activity of telomerase in vitro and in vivo by arsenic trioxide.

Arsenic trioxide (As(2)O(3)) shows a significant therapeutic effect upon acute promyelocytic leukemia (APL) and can induce the apoptosis of NB(4) cells, which attracts scholars' great attention. Especially, the therapeutic effect on solid carcinoma has been paid more close attention to. The present study is to evaluate the effect of As(2)O(3) on human colorectal carcinoma cells (LS-174T cell) and the activity of telomerase in vitro and in vivo. This research made use of the electron microscope, polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), fluorescence-activated cell sorter (FACS), MTT in vitro and in vivo (LS-174T xenograft model of nude mice). With the increasing concentration of As(2)O(3), the ratio of living cells to dead cells decreased significantly, and the IC(50) value was 5.23mumol/L; cells of the experimental groups endured a series of morphological changes similar to the features of apoptosis. Apoptosis curve of FACS pictures appeared after 24h, and the cells showed apoptosis in a time-dependent manner; As(2)O(3) can inhibit the activity of telomerase of the cell extraction, obviously, in a concentration-dependent and time-dependent manner after 24h. As to the inhibition impact of As(2)O(3) on the xenograft model of nude mice in the two indexes, tumor volume and weight, there was a significant difference between As(2)O(3) and the control group; there was no difference between As(2)O(3) and the fluorouracil (5-FU) group; in the group of peritoneal injections of As(2)O(3), the cancer cells connected loosely with each other, nucleus changed markedly, and heterochromatin concentrated under the nucleus membrane. From the in vitro and in vivo experiment, we can see that As(2)O(3) inhibited LS-174T cell growth mainly by inducing cell apoptosis, partly by the inhibition of telomerase activity.     

T cell unresponsiveness correlates with quantitative TCR levels in a transgenic model

Two lines of transgenic mice were developed which differ intheir level of expression of V_ß11. To determine therole of TCR density in tolerance induction, these mice werebred with I-E expressing mice and were investigated for toleranceinduction. T cells expressing V_ß11 at high densityare deleted by negative selection. T cells expressing <10%of the normal TCR receptor density are not subject to negativeselection and were not activated in vitro. There is a correlationin receptor density in both strains of mice with in vitro activation.These findings support the notion that there is a definablequantitative signal threshold which is critical for toleranceinduction.     

T cell-antigen-presenting cell interactions visualized in vivo in a model of antigen-specific inflammation

Videomicroscopy is being used increasingly to characterize the interaction of T cells and antigen-presenting cells (APCs) within lymphatic tissues but has not been reported, to our knowledge, at sites of inflammation. We employed intravital videomicroscopy to study an anterior uveitis model using DO11.10 T cells and ovalbumin (OVA). T cell movement in iris was consistent with a random walk independent of the presence of recognized antigen and had a lateral speed slower than T cells in lymph node. Lingering of T cells adjacent to APCs suggested that they were physically interacting. This apparent contact demonstrated antigen specificity when comparing results from DO11.10 cells with OVA versus bovine serum albumin (BSA) loaded APCs but not when comparing results from OVA-loaded APCs with DO11.10 versus HA clonotype 6.5 T cells. Further studies with this model system should clarify the contribution of T cell-APC communication at a site of inflammation, infection, or immunization.     

Avian scleroderma: evidence for qualitative and quantitative T cell defects.

T cell activation is dependent upon calcium influx and protein kinase C activation, with subsequent lymphocyte proliferation dependent upon IL-2. Abnormalities in T cell proliferation, including abnormal calcium influx and defective protein kinase C activation, have been identified in aged mice and humans and many autoimmune diseases including diabetes, lupus and scleroderma. Since UCD line 200 chickens, which spontaneously develop a scleroderma-like disease, have both thymic defects and a diminished peripheral blood lymphocyte response to IL-2, we have further investigated T cell function in these birds. Interestingly, line 200 T cells respond poorly in vitro to a variety of diversely acting T cell mitogens including concanavalin A, phytohemagglutinin and anti-chicken CD3 monoclonal antibody. Moreover, they do not respond well even to phorbol myristate acetate in conjunction with ionomycin. Addition of exogenous IL-2-containing supernatant concurrently with mitogenic stimulation also had no significant effect. Analysis of intracellular free calcium demonstrated that the lymphocytes from diseased birds had a reduced influx of calcium (or release for intracellular stores) following stimulation. These data clearly reflect a unique defect in T cell activation associated with avian scleroderma. Analysis of chicken CD3, CD4 and CD8 expression revealed a 39% decrease in peripheral blood CD4+ cells in scleroderma birds, although this decrease was not sufficient to explain the 80-90% decrease observed in proliferation assays and calcium influx. Our data support the hypothesis that avian scleroderma is mediated via abnormal function of lymphocyte co-stimulatory molecules or intracellular calcium regulators.     

T cells in hypersensitivity pneumonitis: effects of in vivo depletion of T cells in a mouse model.

C57BL/6 mice were given intranasal instillation of optimal doses of the actinomycete Faeni rectivirgula 150 micrograms/mouse 3 days/wk), an important offending agent causing hypersensitivity pneumonitis. This instillation was associated with a very significant increase in the lung weight of the mice and also a large increase (10-fold) in the number of cells recovered from the bronchoalveolar lavage (BAL) of instilled mice. Also, this instillation was associated with a very significant fibrosis at 4 and 8 wk (2-fold increase in hydroxyproline levels in the lungs). We determined the effect of depleting certain T-cell subsets on the progression of this inflammatory disease. Elimination of the L3T4 subset did not significantly affect the increase in the lung index, the lung cellular influx, or its profile. Fibrosis was also unaffected by this depletion of L3T4+ cells. Similarly, depletion of Lyt2+ (CD8+) cells did not lead to significant changes in these disease parameters. Depletion of all T cells (Thyl+) was also ineffective at modifying the number of infiltrating cells and the lung index score. However, identification of cell types in BAL showed that mice depleted of Thyl+ cells had a cellular influx that was almost exclusively neutrophilic throughout the instillation period, whereas control mice developed only a transient neutrophilic response to F. rectivirgula instillation, which was replaced by a recruitment of mononuclear cells, mostly macrophages. Also, depletion of Thyl+ cells before and during F. rectivirgula challenge had no effect on tumor necrosis factor-alpha levels in the BAL of treated mice (63 +/- 13 U/ml in anti-Thy1. 2 antibodies treated versus 52 +/- 10 U/ml in the BAL of control mice given F. rectivirgula).(ABSTRACT TRUNCATED AT 250 WORDS)     

T cell requirements for the expression of the lipopolysaccharide adjuvant effect in vivo: evidence for a T cell-dependent and a T cell-independent mode of action.

The in vivo adjuvant effect of lipopolysaccharide (LPS) in mice was investigated with the soluble synthetic polypeptide antigen (T, G)-A--L, the antibody response to which is determined by the Ir-1A gene. With this specific antigen it can be demonstrated that the LPS adjuvant effect has the following modes of action: a) a T cell-dependent enhancement of primary and secondary IgM antibody response; b) a T cell-dependent enhancement of IgG secondary andibody response; and c) a T cell-dependent induction of switchover from IgM to IgG andibody in some strains of Ir-1A low responders. Although T cells are necessary for some aspects of the adjuvant effect, these data do not distinguish between a mechanism involving a direct interaction between LPS and T cells or a direct interaction of LPS and B cells with a general requirement for T cells for expression of IgG antibody.     

Unheated serum reagin test as a quantitative test for syphilis.

The unheated serum reagin (USR) test, a standard qualitative test for syphilis, was evaluated for use as a quantitative test for the serodiagnosis of syphilis and as a means of determining the effectiveness of treatment. Tests were performed on 664 syphilitic sera from treated and untreated patients in the primary, secondary, and latent stages of the disease. Also included were 95 false-positive sera and 36 sera from presumably normal donors. The Venereal Disease Research Laboratory slide and rapid plasma reagin 18-mm circle card tests were used as a basis for comparison with the USR test. Results of the USR quantitative test paralleled the Venereal Disease Research Laboratory and rapid plasma reagin card quantitative tests with approximately 84% agreement. The advantages of the USR test make the findings of this study significant in that: (i) less technical manipulation is required to perform the USR test than the Venereal Disease Research Laboratory slide test; (ii) no labor cost is incurred for daily Venereal Disease Research Laboratory antigen preparation; and (iii) the USR antigen is less expensive than the rapid plasma reagin card tests antigen.     

Methods for quantitative evaluation of alveolar structure during in vivo microscopy

In vivo fibred confocal laser scanning microscopy allows an evaluation of differences in alveolar mechanics between healthy and acutely injured lungs during mechanical ventilation. The aim of this study was to develop new methods for a quantitative analysis of microscopic images in a murine model of experimental acute lung injury (ALI) and to assess the methods' portability to a large animal model. Differences observed in ALI compared to healthy lungs were: reduction of air-filled areas, increase of heterogeneity and increase of shape irregularity. Three indices were developed: the volume air index (VAI) applies an integral over specific signal intensities, the heterogeneity index (HI) and the Heywood circularity index (CI) comprise variances in size and shape of alveolar structures. The differences between healthy and ALI conditions were found to be significant for all of the used indices (VAI: 0.648 vs. 0.443 (p < 0.05), HI: 0.852 vs. 1.348 (p < 0.001) and CI: 1.56 vs. 1.66 (p < 0.001)). The portability of these algorithms to a porcine model was confirmed reaching similar results (VAI: 0.50 vs. 0.35, p < 0.05; HI: 0.62 vs. 1.83, p < 0.05; CI: 1.56 vs. 1.63, p < 0.001). VAI, HI and CI may help to quantify microscopic images of changes in alveolar structure after experimental ALI.     

Cultured T lymphocytes cytotoxic for a syngeneic lymphoma: derivation in Con A-conditioned medium and in vivo activity.

Cultures of murine T lymphocytes with cytotoxic activity towards syngeneic RBL-5 lymphoma cells were obtained from spleen cells of immunized animals after co-culture in vivo with irradiated RBL-5 cells. At different times after initiation, these mixed tumour-lymphocyte cultures (MTLC) were multiplied by transfer to conditioned medium (CM) containing T cell growth factor (TCGF) activity, produced by the stimulation of rat spleen cells with Con A. The effect of residual Con A was investigated by the addition of specific blocking sugar, alpha-methyl mannoside (alpha MM), to the CM in some experiments. This procedure did not reduce the growth potential of the cells, and resulted in a dramatic increase in the cytotoxic activity of the cultures as measured by a 4-hr 51Cr-release assay. The cultures multiplied 1 X 10(3)-fold over a 3-week period with retention of cytotoxicity for RBL-5 cells at levels up to 70-fold greater than those of the MTLCs from which they were derived. The cultured cells, when injected i.p. together with RBL-5 cells into normal mice, were shown to mediate a significant prolongation of the survival of the treated animals. This effect was, however, less dramatic than had been expected from the in vitro results. It would therefore appear that, while cells grown in tissue culture using Con A-conditioned medium may fulfill some theoretical requirements for the immunotherapy of experimental tumours, other factor(s) are required for full protection.     

A quantitative evaluation for peripheral respiratory chemosensitivities by the withdrawal test in man.

Ten healthy subjects were tested for their peripheral respiratory chemosensitivities by the withdrawal technique two times on separate days. Hypoxic hypercapnia of PET, O2 75, 65 AND 55 mmHg with PET, CO2, 5 mmHg higher than the control level was replaced by 100% O2 two times with spontaneous respiration. Then, breath-by-breath depression calculated in minute ventilation (delta V) was observed during the period 5-20 sec after the first O2 inhalation. The results were analyzed by the linear relationship between PET, O2 and 1n delta V, and PaO2 and 1n delta V. Delta V at P02 50 mmHg, delta V50, was 9.09 +/- 6.81 liters/min (mean +/- SD) in PET, O2-1n delta V analysis and 9.22 +/- 7.46 liters/min in PaO2-1n delta V analysis, respectively. The averaged day to day variation of delta V50 expressed by SE in % was 5.3% in PET, O2-1n delta V analysis and 11.5% in PaO2-1n delta V analysis, respectively.     

Dendritic cell elimination as an assay of cytotoxic T lymphocyte activity in vivo

We show in this paper that the survival of antigen-loaded dendritic cells in vivo may be used as a sensitive readout of CTL activity. We have previously shown that dendritic cells labeled with the fluorescent dye CFSE and injected sub-cutaneously into mice migrate spontaneously to the draining lymph node where they persist for several days. In the presence of effector CTL responses, dendritic cells loaded with specific antigen rapidly disappear from the draining lymph node. In this paper we extend the above observations and set up a simple and sensitive method to reveal CTL activity in individual mice in vivo. Dendritic cells were labeled with two different fluorochromes, loaded with antigen or left untreated, and mixed together before injection into mice. We show that only the dendritic cells loaded with specific antigen were cleared from the draining lymph node, while dendritic cells not loaded with antigen remained unaffected. Cytotoxic responses generated by immunization with peptide-loaded dendritic cells, or by infection with influenza virus, could be revealed using this method. Comparison of the differential survival of dendritic cells populations mixed together also allowed us to accurately evaluate the disappearance of dendritic cells, irrespective of variability in the injection site and other parameters. Given the ability of dendritic cells to efficiently take up and present complex antigens, nucleic acids and apoptotic bodies, this method may also allow the evaluation of cytotoxic activity against antigens that are not characterized in terms of peptide epitopes.     

Presentation of superantigen by human T cell clones: a model of T-T cell interaction.

Superantigens (SAg) interact with T lymphocytes bearing particular V beta sequences as part of their T cell receptor (TcR). The interaction, however, requires the presence of major histocompatibility complex (MHC) class II molecules on antigen-presenting cell (APC). In peculiar circumstances, MHC class II+ T cell clones (TCC) have been shown to present peptides and selected antigens interacting with antigen-specific TCC in the absence of APC. In this report we studied the capacity of SAg to mediate a T-T cell interaction, investigating the TCC ability to present a panel of staphylococcal enteroxins (SE) independently of the presence of added APC. Upon exposure to a broad range of SE concentrations, MHC class II+ TCC showed an intense proliferative response even in the absence of professional APC. Diverse SE optimally stimulated responder TCC at different concentrations. The proliferation was inhibited by anti-DR monoclonal antibodies, both in the presence and in the absence of APC. The SE activation of TCC in the absence of APC induced the same series of phenotypic variations as that observed following the TCC stimulation with APC. Irradiated TCC efficiently presented membrane-bound SE to responder TCC as well as professional APC. These results show that a single cell of a given clone effectively presents the SE to other cells of the same clone, and provide evidence that SAg can efficiently mediate T-T cell interaction. In addition, the possibility also exists that one cell of the clone can actually undergo an auto-stimulation via SAg-mediated interactions between its own TcR and MHC class II molecule. It has recently been suggested that the V beta-selective depletion of T cells observed in acquired immunodeficiency syndrome (AIDS) patients might be a consequence of the interaction between a human immunodeficiency virus (HIV)-encoded SAg and T cells expressing a SAg complementary V beta. We suggest that the hypothesized HIV-encoded SAg might mediate T-T cell interactions that could play a relevant role in the V beta-selective depletion of T lymphocytes observed in HIV-infected patients.     

Visualizing T cell migration in vivo

Ever since the realization that T lymphocytes are key players in the defense against pathogens and tumors, a major aim of immunologists has been to understand the relationship between the functional and migratory properties of antigen-specific T cells. The current paradigm proposes that T cells follow organ-specific trafficking pathways to exit from blood into the extravascular compartment. T cell homing is regulated at the level of adhesion molecules and chemokine receptors, whose expression is linked tightly to the differentiation state of the cell. Na?ve T lymphocytes follow relatively uniform recirculation routes through secondary lymphoid organs, the molecular cues of which are fairly well understood. As effector and memory T cells must be capable of reaching virtually any site in the body, their migratory behavior is considerably more heterogeneous. During the past few years, innovative approaches for tracking T cells in vivo have emerged. Here, we review recent technical developments in experimental methods for the visualization of T cells both at the population and single cell level in vivo, and discuss what these methods have taught us about T cell trafficking.     

T细胞运载金纳米粒子活体成像的初步实验

目的为直观评价过继免疫疗法的疗效,尝试用电穿孔法将显像剂标记的金纳米粒子（AuNPs）导入T细胞,再对T细胞进行活体成像。方法3只雄性BALB/C-nu/nu裸鼠,6周龄,体质量18～22 g。①以氨羧络合剂（DTPA或DOTA）对AuNPs的表面进行改性,再用聚乙二醇（PEG）修饰,最后对AuNPs进行111In或64Cu标记。②通过电穿孔法将AuNPs导入正常人T细胞。③将含111In标记AuNPs的T细胞直接注射到裸鼠1肺内,采用微型单光子发射计算机断层成像术（micro-SPECT）/CT对其活体成像。再以微型正电子发射断层成像术（micro-PET）/CT对含有64Cu标记AuNPs的T细胞于裸鼠2及64Cu标记AuNPs于裸鼠3体内生物学分布进行活体成像。结果电穿孔法将111In标记的AuNPs导入T细胞后直接注射入裸鼠1肺内,以micro-SPECT/CT对其成像,结果为阳性,裸鼠1右肺内见高摄取区。用正电子核素64Cu标记AuNPs,同时尝试各种电转条件以进一步提高转染效率（2.3×105AuNPs/cell）和细胞存活率（65.4%）,经尾静脉注射入裸鼠2体内,micro-PET/CT于注射后立刻成像、注射后2 h及18 h分别成像,清晰地显示含有64Cu标记AuNPs的T细胞在正常裸鼠2体内的动态生物学分布。作为对照,同时对64Cu标记的AuNPs本身于裸鼠3体内活体成像,发现含有AuNPs的T细胞与AuNPs本身的生物学分布显著不同。结论初步研究表明运载显像剂标记AuNPs的T细胞活体成像是可以实现的。     

Methyl isocyanate: an evaluation of in vivo cytogenetic activity

The ability of inhaled methyl isocyanate (MIC) to induce genotoxic and cytotoxic damage in vivo was evaluated by assessing the induction of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) in bone marrow metaphase cells, the induction of micronuclei in polychromatic erythrocytes (MN-PCEs), and the inhibition of bone marrow cellular proliferation and erythropoiesis. B6C3F1 mice were exposed to MIC by two exposure regiments: in two experiments, male mice only were exposed to 3, 10, and 30 ppm for 2 hr; in four experiments, male and female mice were exposed to 1 and 3 ppm (in one experiment, to 6 ppm, also), 6 hr per day for 4 consecutive days. The various cytogenetic endpoints were analyzed in bone marrow and peripheral blood (4-day exposure regimen only) samples taken from bromodeoxyuridine tablet-implanted animals killed 11 to 22 hr after cessation of the exposure to MIC. Exposure to MIC for 2 hr induced a significant delay in cellular proliferation but did not induce a significant increase in CAs, SCEs (evaluated at 3 and 10 ppm, only) or in bone marrow MN-PCEs. Also, this exposure regimen did not inhibit the rate of erythropoiesis. Following exposure to MIC for 4 days, a weak but significant increase in CAs and SCEs was observed in male (in one experiment) and in female (in two experiments) mice. The induction was especially apparent in the single experiment in which mice were exposed to 6 ppm MIC. At this concentration, a significant increase in MN-PCEs in peripheral blood was observed in male but not female mice. Delay in bone marrow cell proliferation was observed in male mice beginning at 3 ppm and in female mice at 6 ppm. The 4-day exposure regimen resulted also in a depressed rate of erythropoiesis, with male mice appearing to exhibit greater depression than female mice. The results demonstrate that exposure to MIC by inhalation results in bone marrow damage, indicating the systemic genotoxic/cytotoxic activity of MIC and/or reactive metabolites.     

Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of beta1 and alpha6 integrins and are distinct from SP-KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays. When in vitro assays were used to measure total cell output of human SP-KCs and integrin-bright keratinocytes, we could not document their superior long-term proliferative activity versus unfractionated keratinocytes. To further assess the KSC characteristics in SP-KCs and integrin-bright keratinocytes, we used an in vivo competitive repopulation assay in which bioengineered human epidermis containing competing keratinocyte populations with different human major histocompatibility (MHC) class I antigens were grafted onto immunocompromised mice, and the intrinsic MHC class I antigens are used to quantify expansion of competing populations. In these in vivo studies, human SP-KCs showed little competitive expansion in vivo and were not enriched for KSCs. In contrast, keratinocytes expressing elevated levels of alpha6 integrin and low levels of CD71 (alpha6-bright/CD71-dim) expanded over 200-fold during the 33-week in vivo study. These results definitively demonstrate that human alpha6-bright/CD71-dim keratinocytes are enriched with KSCs, whereas SP-KCs are not.     

Peak discrimination as a method for quantitative evaluation of neural activity by computer

A computer method (PDP-12) for the quantification of neural activity in single- as well as few-fibre preparations of nerves was devised and tested on function-generator signals and on neurograms from baro- and chemoreceptor fibres of the carotid sinus nerve of rabbits. The principle of the program was to search continuously for maxima and minima of the electrogram regardless of the absolute voltage range in which they occurred. Action potentials were then distinguished from noise by comparing these maxima with amplitude discriminators. The latter were derived from the previous analysis of noise in selected neurograms and in recordings during activity-free intervals of the normal electrogram or temporary cold block of the preparation. From peaks accepted as action potentials the program computed total mean frequency, amplitude histogram and interval histogram as standard outputs. The method permitted one to determine in few-fibre preparations not only total activity but also response characteristics of different fibre components. However, owing to principal limitations shown to be inherent in such techniques, the exact quantification of neurograms from several fibres became impossible if the mean frequency—depending on the type of activity—exceeded several hundred to about 1000 impulses/s. By systematic variation of program parameters and type and rate of neural activity, the performance characteristics of the program as well as certain general properties of single-, few- and multi-fibre neurograms were evaluated.