Expansion of mature thymocyte subsets before emigration to the periphery

A small population of DNA-synthesizing mature thymocytes could be defined by analyzing cell surface markers and 5-bromo-2'-deoxyuridine (BrdUrd) labeling by four-color cytofluorometry. These cells have a completely mature phenotype (CD4- CD8+ or CD4+ CD8- TCR(high), HSA-, Qa-2(high)) and expand only weakly after BrdUrd incorporation. They recovered immediately in total number and in DNA synthesis rate after treatment with the antimitotic drug demecolcin, thus much faster than immature CD4+ CD8+ thymocytes. These data demonstrate the existence of a late intrathymic expansion phase, independent of that of developing CD4+ CD8+ immature cells, and involving phenotypically mature cells renewed each day. In mixed chimeras prepared by transfer of bone marrow and lymph node cells into RAG-2(-/-) mice, all cycling mature thymocytes were bone marrow derived. They are thus produced in situ and do not correspond to peripheral T cells reentering the thymus. Double FITC/BrdUrd detection showed that a high proportion (10-20%) of recent thymic emigrants were BrdUrd+ just postcycling cells and that around 50% of cycling mature thymocytes are just ready to emigrate to the periphery in the few hours after DNA synthesis. The late intrathymic expansion phase demonstrated here increases the daily thymic cell export by at least 30%. It could play a role in the adjustment of the T cell repertoire before emigration and in the regulation of the thymic cell output into the peripheral T cell pool.     

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.     

Responses to human rhinovirus in CD45 T cell subsets isolated from tonsil

Isotypes of CD45 have been used extensively as markers of memory and naive populations of T cells in peripheral blood. In this study, T cells were isolated from human tonsil and their proliferative response against human rhinovirus was measured. Unexpectedly, equivalent responses were found among the CD4+CD45RA+ and CD4+CD45RO+ populations of T cells. This response requires MHC class II-positive antigen-presenting cells. The time course of the T cell response in vitro was that of a classical recall response, and no proliferative response to the virus could be detected in human cord blood. These results suggest that tonsils contain a significant population of CD45RA+ memory cells. The presence of this population may reflect ongoing stimulation with this common infectious agent, and the anatomical location of the T cells within the major lymphoid organ draining the naso-pharyngeal epithelial surface.     

Altered cellular calcium responsiveness to insulin in normal and hypertensive pregnancy

OBJECTIVE: To investigate the glucose-independent calcium-related effects of insulin from subjects with normal and hypertensive pregnancies. METHOD: We used lndo-l fluorescence spectroscopy to measure cytosolic free calcium levels (Cai) in peripheral blood mononuclear cells (PBM) from 17 women (aged 20-40 years), six nonpregnant controls (NPC), five pregnant normotensive (PNT) women and six pregnant hypertensive (PHT) women, before and 5, 30, 60, 120 and 180 min after in vitro incubation with 200 microU/ml insulin. RESULTS: Basal Cai levels were significantly higher in PHT women (175.2 +/- 18.8 nmol/l) than they were in NPC women (122.8 +/- 2.8 nmol/l) and PNT women (123.9 +/- 3.5 nmol/l). The initial insulin-induced rise in Cai was similar in NPC (delta Cai 13.5 +/- 5.6 nmol/l) and PNT women (delta Cai 14.6 +/- 3.7 nmol/l), but appeared blunted in PHT women (delta Cai 8.2 +/- 3.5 nmol/l), and, for all pregnant subjects, was closely and inversely related to basal Cai. Over time, in PNT women, delta Cai did not increase from the initial response (maximal delta Cai 20.5 +/- 2.3 nmol/l) compared to NPC. The total cellular calcium response to insulin was also blunted in PNT women (the area under the calcium-responses curve was 86 +/- 3.4 versus 97.4 +/- 6.5 nmol/l), but was excessive in PHT women (115.5 +/- 6 nmol/l, P = 0.05). CONCLUSIONS: Hypertension in pregnancy is associated with excess Cai, insulin raises Cai in PBM, and different alterations of Cai responsiveness to insulin occur both in normal and in hypertensive pregnancy. These cellular calcium alterations may help to explain altered tissue responsiveness to insulin and other hormones in pregnancy.     

Altered growth hormone and prolactin responsiveness to TRH in the infant rat

12-day-old female and male pups were killed 10 min after the injection of either saline or thyrotropin releasing hormone (TRH), and plasma growth hormone (GH) and prolactin (PRL) levels were measured by radioimmunoassay (RIA). At all doses used (0.15, 0.3, 0.6 and 1.5 mug/100 g b.w.i.p.), TRH induced a significant, although not dose-related, increase in plasma GH levels, but was effective in releasing PRL only at the greatest dose level (1.5 mug/100 g b.w.). The GH-releasing effect of TRH was even more evident in 12-day-old pups subjected to central sympathectomy of 6-hydroxydopamine (6-OHDA, 60 mug/10 mul intraventricular route) 1 week before; in these animals, TRH was ineffective in releasing PRL even at the greatest dose level (1.5 mug/100 g b.w.). In pups pretreated with 6-OHDA, the GH-lowering effect of insulin hypoglycemia or cold exposure was markedly reduced, while the PRL responses were unmodified. Baseline plasma PRL levels were markedly increased following 6-OHDA administration. It is proposed that in the infant rat the greater GH than PRL responsiveness to TRH, which opposed the pattern of response present in the adult animal, may be due to the existence of a 'physiologic' functional disconnection between the central nervous system (CNS) and the anterior pituitary (AP). Results obtained following central sympathectomy by 6-OHDA, which further disrupted CNS-AP links, substantiate this view.     

Altered growth and lack of responsiveness to angiotensin II in aortic vascular smooth muscle cells from cirrhotic rats

BACKGROUND & AIMS: In cirrhosis, increased amounts of circulating hormones such as angiotensin II may induce vascular tone changes and alter vascular smooth muscle cell (VSMC) function and growth. The aim of this study was to investigate the growth of aortic VSMCs from cirrhotic rats with or without the addition of angiotensin II and to determine whether angiotensin II binding was preserved in cirrhotic VSMCs. METHODS: Cirrhosis was induced by bile duct ligation. Cell growth was studied in cultured aortic VSMCs at passage levels between 4 and 16 by determining cell number and protein synthesis. RESULTS: Proliferation rates of cirrhotic VSMCs were lower than those of control VSMCs. The addition of angiotensin II to control VSMCs caused an increase in cell proliferation and protein synthesis. This increase was not observed in cirrhotic cells. There were more angiotensin II receptors in cirrhotic than in control VSMCs, but no significant changes in affinities were found. Angiotensin II-stimulated protein synthesis was dependent on protein kinase C activity and increased intracellular Ca2+ concentrations. CONCLUSIONS: This study shows abnormalities in growth characteristics and responsiveness to angiotensin II of cultured aortic VSMCs from rats with cirrhosis.     

CD56bright natural killer cell subsets: characterization of distinct functional responses to interleukin-2 and the c-kit ligand

Horizontal transmission of Salmonella enteritidis and the effect of airflow on spreading were examined in 80 5-wk-old chickens divided into five groups. Sixteen chickens in each group were placed in four cages in a row separated by wire. One among four chickens placed in a cage at the downwind end of the row was inoculated orally with 10(9) colony-forming units of S. enteritidis. Cecal droppings, drinking water, and feed were cultured every day. Horizontal transmission was rapid in the row with low air velocity but slow in the row with high air velocity. However, in another experiment, where the inoculated chicken was situated in a cage upstream in the airflow, horizontal transmission was equally rapid whether the airflow was rapid or slow. Contamination of feed and water never preceded the appearance of positive cecal droppings. These findings suggest that the rapidity of horizontal transmission of S. enteritidis may be affected by airflow patterns.     

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody

This study investigated the response of different CD5- B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human mu chain antibody (a-mu-Ab). The different CD5- B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5+ B cells and subsequently fractionated on Percoll density gradients. While resting CD5+ B cells proliferated and produced IgM molecules in response to a-mu-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5- B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5+ B cells had the typical phenotype of mantle zone B cells (IgM+ IgD+ CD39+ CD38- CD10- CDw75dim), whereas resting CD5- B cells were CD38- CD39- CD10- CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38+ CD10+ CD39- CDw75bright, IgG+) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5- B cells. However, some of the data collected suggest possible relationships between CD5- B cells and germinal center cells. The CD5- B cells isolated from the 50% Percoll fraction proliferated in response to a-mu-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5+ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5+ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5- B cells from the 50% Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5- B cells did not express CD5. The CD5- B cells from the 50% Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polysaccharides from Agaricus blazei stimulate lymphocyte T-cell subsets in mice

Subset analysis of splenic lymphocytes using flow cytometry showed that the percentages of Thy1.2-(pan T-cells), L3T4-(CD4, helper T-cells), and Lyt2-(CD8, cytotoxic T-cells) positive cell populations were significantly increased in mice orally administered a hot water-soluble fraction from Agaricus blazei as compared with mice treated only with saline. 13C-NMR data indicates that the main component in the active polysaccharide is the complex of alpha-1,6- and alpha-1,4-glucan, which had already been shown to have anti-tumor activity against Sarcoma 180. It seems that the polysaccharide from Agaricus blazei may be an effective prophylactic, protecting humans against cancer by stimulating lymphocytes such as cytotoxic T-cells.     

CD95 predicts responsiveness to tretinoin in acute promyelocytic leukemia

We describe a predictive marker (CD95) for the responsiveness to tretinoin (RA) in acute promyelocytic leukemia (APL). Functional CD95 expression during RA treatment have been observed only in those patients who responded to RA. Expression of CD95 (Fas antigen), which plays a major role in apoptosis, was determined by fluorescence activated cell sorter (FACS) analysis. APL cases in which no enhancement of CD95 expression was observed showed no response to RA and did not obtain complete remission. We propose that CD95 can predict the clinical response to RA probably due to differentiation.     

Expression and functional analysis of glycosyl-phosphatidyl inositol-linked CD46 in transgenic mice

OBJECTIVE: To determine whether a high level of hopelessness after treatment of a depressive episode is an indicator of a history of lifetime suicide attempts in older people. DESIGN: Groups of suicide attempters, suicidal ideators, and nonsuicidal patients were compared via analysis of variance with respect to levels of hopelessness, depression, anxiety, and global functioning before and after treatment of depression. SETTING: An outpatient research clinic providing two treatment protocols of late-life mood disorders. PARTICIPANTS: A total of 107 consecutive outpatients (mean age 67) with major depression who responded to treatment. MEASUREMENTS: Levels of hopelessness, severity of depression, anxiety, and global functioning were compared across the three groups at the beginning of treatment and at remission. RESULTS: After remission there were no differences between the three groups in depression severity, anxiety, and global functioning. Hopelessness remained significantly higher in the attempter group than among ideators or nonsuicidal patients. CONCLUSIONS: Suicide attempts, the most important risk factor for subsequent suicide, are associated with persistent, high levels of hopelessness following remission of depression in late-life patients. These findings suggest that treatments designed specifically to lower hopelessness (such as cognitive, behavioral or interpersonal therapy) may be effective in reducing suicide risk.     

The functional paradox of CD43 in leukocyte recruitment: a study using CD43-deficient mice

OBJECTIVE: To determine the variable most closely related to symptomatic relief of osteoarthritis (OA) of the knee in response to a weight control program. METHODS: Twenty-two patients diagnosed with knee OA whose body mass index (BMI) was more than 26.4 were treated with a low calorie diet, an appetite suppressant, and nonsteroidal antiinflammatory drugs for 6 weeks. The patients were instructed to follow a walking program. We analyzed BMI, percent body fat, the average number of steps per day by pedometer, and the metabolic correlates of obesity (blood pressure, fasting blood serum glucose, total cholesterol, triglycerides, and serum insulin) at the beginning and end of therapy. The correlation between the change in each variable and the remission score (delta score) using the Severity Index of Lequesne, et al was evaluated. RESULTS: Delta score of knee OA was more strongly associated with reduction in percent body fat (p= 0.0013, r=0.62) than other variables. Significant correlation was also observed between the number of steps per day and delta score (p=0.0031, r=-0.58). No other variable, including weight loss, was significantly correlated with delta score. There was a significant correlation between delta percent body fat and the number of steps per day (p=0.012, r=-0.62). CONCLUSION: In a weight control program, decreasing body fat and increasing physical activity are more important than body weight loss or decreasing other indices of obesity in producing symptomatic relief of knee OA, although there is not necessarily a cause and effect relationship between body fat and OA score.     

Skewing of TCR V genes to CD4+ and CD8+ subsets of T lymphocytes is not determined by amino acid composition of CDR3

TCR V genes show differing expression patterns, termed skewing, in CD4+ and CD8+ subsets of T lymphocytes. To determine which elements of the TCR V regions contribute to these observed TCR V gene skewing patterns, we have performed an in-depth analysis, taking advantage of RT-PCR and DNA sequencing, which was focused on the multi-member TCRBV6 gene family. These studies allowed us to evaluate the contributions of the various elements, that constitute the TCR beta chain variable region, to the observed TCR V gene skewing patterns. The results of these analyses revealed that within the TCRBV6 family individual members exhibited differing skewing patterns, i.e. TCRB6S7 was significantly skewed towards the CD4+ T cell subset, whereas TCRBV6S5 was significantly skewed towards the CD8+ subset. Scrutiny of the usage of TCRBV6 family members in combination with TCRBJ gene usage and amino acid composition of CDR3 did not reveal obvious structural characteristics which would explain the differing skewing patterns between TCRBV6S7 and TCRBV6S5. Further examination of these TCR V regions showed that the CDR1 and 2 regions within these TCRBV elements were composed of different amino acids. These observations suggests that these components contribute to the observed TCR V gene skewing patterns.     

Altered T-lymphocyte responsiveness to polyclonal cell activators is responsible for liver cell necrosis in alcohol-fed rats

The role of T-cell activation in alcoholic liver disease was investigated in rats fed alcohol and subsequently exposed to concanavalin A (Con A). Following Con A injection (20 mg/kg body weight), greater increases in liver-to-body weight ratio and ALT levels were observed at 12 and 24 hr in rats fed ethanol, compared with control rats fed sucrose. Furthermore, increases in serum interleukin-6 and tumor necrosis factor-alpha levels were noted in ethanol-fed rats, with maximal levels detected at 4 hr declining thereafter, but remaining above control levels at 24 hr. Analysis of T-cell subpopulations showed an increased percentage of CD4+, CD5+, and CD8+ T cells in blood from all groups, but not in liver perfusate. In contrast, a significant increase in the percentage of activated CD25+ T cells was detected in both blood and liver perfusate from rats fed ethanol even 24 hr after Con A injection. When CD4+ and CD8+ T cells from liver perfusate were cultured in the absence or presence of Con A, an increase in interleukin-6 and tumor necrosis factor-alpha production in supernatants was observed in ethanol-fed rats. In cultures stimulated with Con A, a 2- to 8-fold increase in cytokine production was detected, with intrahepatic CD4+ T cells being the major source. Immunohistological analysis revealed infiltration of CD4+ T cells around portal vein and central vein areas associated with fatty liver and severe hepatic necrosis. The results suggest that alcohol consumption induced a dysregulated T-cell population that mediated hepatic necrosis following polyclonal activation with Con A.     

The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte

T cell development is regulated at two major control points where maturation, proliferation, and antigen receptor gene rearrangement are coordinated. Progression through these developmental control points is dependent upon the expression of different forms of the T cell receptor. Here we show that the MAP kinase cascade is a regulator of the differentiation of immature thymocytes from double-negative to double-positive cell, most probably acting as a transducer of pre-T cell receptor signaling. Furthermore, this study demonstrates the use of retrovirus-mediated gene transfer in fetal thymic organ culture in the analysis of thymic development in mutant mice, an alternative to transgenesis by oocyte injection.     

Expression of a CD3 epsilon transgene in CD3 epsilon(null) mice does not restore CD3 gamma and delta expression but efficiently rescues T cell development from a subpopulation of prothymocytes

The TCR-associated CD3 complex consists of four subunits, i.e. CD3 gamma, delta, epsilon and zeta, which are expressed very early in T cell development prior to the expression of the TCR and the pre-TCR alpha chain. It is unclear whether the expression of each CD3 protein is independent of, or is influenced by, other CD3 subunits. To study whether CD3 epsilon regulates expression of CD3 gamma and delta genes, we generated a strain of CD3 epsilon-deficient mice termed CD3 epsilon(delta P/delta P) (epsilon(delta P)), in which the promoter of CD3E was disrupted, and subsequently reconstituted these mice with a CD3 epsilon transgene. In the epsilon(delta P) mice, T cell development is arrested at the double-negative stage and targeting the CD3 epsilon gene caused severe inhibition of CD3 gamma and delta gene expression. Introduction of the CD3 epsilon transgene did not restore CD3 gamma and delta expression. However, a very small fraction of prothymocytes that expressed CD3 gamma and delta was rescued upon reconstitution of the CD3 epsilon transgene. Remarkably, this rescue led to a very efficient differentiation and maturation of thymocytes, resulting in a significant T cell population in the periphery. These results demonstrate that CD3 epsilon does not regulate expression of CD3 gamma and delta genes, and underscore the capacity of each prothymocyte to give rise to a large number of mature peripheral T cells.     

Regulation of CD27 expression on subsets of mature T-lymphocytes

CD27, a lymphocyte-specific member of the TNF/NGF-R family, is expressed on the majority of peripheral blood T cells. Activation of T cells via TCR/CD3 induces high CD27 surface expression and the release of a soluble extracellular part of the molecule. After prolonged activation in vitro, CD27 becomes gradually switched off. There is evidence that also in vivo, CD4+ cells that have persistently been stimulated by Ag, accumulate within the CD45RA-CD27- subset. In addition, an increase of CD27- T cells has been observed under certain immunopathologic conditions and during aging. This study was undertaken to analyze the regulation of CD27 on different T-cell subsets and to determine whether the loss of CD27 expression is an irreversible event and may thereby mark T-cell differentiation. In agreement with earlier findings, all CD4+CD45RA+CD45RO- T cells were found to express CD27, whereas a small fraction of the CD4+CD45RA-CD45RO+ subset lacks the molecule. In contrast, within the CD8+ compartment CD27- subsets were found both in the CD45RA+ and CD45RA- subpopulations. After stimulation with CD3 mAb, both CD27 membrane expression and release was equally up-regulated in CD4+ and CD8+ subpopulations. This stimulus, however, provoked a strikingly predominant up-regulation of membrane CD27 on CD45RO- cells as compared with CD45RA- cells. On CD4+CD45RA-CD27- T cells and long-term grown CD45RA-CD27- TLC, CD27 expression could not be reinduced after stimulation of the TCR/CD3 complex, neither at the protein nor at the mRNA level. Comparison of CD27 expression with its structural homologue FAS/APO-1 showed that down-regulation after prolonged activation is not a general feature of TNF/NGF-R family members. The CD27 ligand was recently identified and was shown to give a co-stimulatory signal to PHA-activated T cells. The restricted up-regulation of CD27 on CD45RA+ cells after T-cell stimulation may point at a discrete role of CD27-CD27 ligand interaction during transition of CD45RO- to CD45RA- T cells. In addition, the CD27 negative phenotype seems a stable reflection of differentiation rather than of activation.