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PubMed1982

Selection of the T cell repertoire during ontogeny: limiting dilution analysis.

The relative frequencies of cytotoxic T lymphocyte precursors (CTL-P) specific for minor histocompatibility antigens that are restricted to either k or b major histocompatibility complex antigens in normal B6 mice and in B6 leads to (B6 x CBA)F1 radiation chimeras were estimated. The ratio of k-restricted to b-restricted CTL-P determined for the radiation chimeras served as a base value to which the ratio of k: b-restricted CTL from normal B6 mice can be compared. The frequencies of allorestricted CTL-P in B6 mice were determined after the B6 cells had been depleted of k-reactive cells by filtration through irradiated B6.H-2k mice. Lymphocytes from immunized animals were used to obtain frequency estimates of CTL-P specific for minor H antigens. The cultures contained nu/nu spleen cells syngeneic to the responder, but no exogenous interleukin 2 (T cell growth factor) since a high degree of nonspecific killing was found in IL2-supplemented cultures. It was found that the CTL-P frequency of B6 mice to BALB.B antigens in the lymph node cells of normal or immune B6 mice were 1 in 300 000 and 1 in 8000, respectively. In B6 leads to (B6 x CBA)F1 radiation chimeras, the ratio of BALB.B/BALB.K-specific CTL-P was 3:1; this ratio was 17:1 in normal B6 mice. Twenty-five percent of CTL clones from normal B6 mice lysing BALB.K target also lysed BALB.B target whereas less than 1% of CTL clones lytic on BALB.K target from the chimeras showed this cross-reaction The results are compatible with the idea of complete positive selection of self MHC-restricted CTL-P in the absence of priming effects by foreign antigen.

Animals↗
PubMed1988

Inverse correlation between the utilization of an idiotype in specific immune responses and its representation in pre-immune "natural" antibodies.

Previously we have characterized an idiotype (Id) that accounts for half of all specific anti-dextran B512 (Dex) antibodies in C57BL/6 mice. BALB/c mice produce the same Id in normal, pre-immune sera but fail to use it in antibody responses to Dex, although Id+ anti-Dex antibodies can be induced in this strain by anti-Id immunization. By limiting dilution analysis of B cell clonal precursors, we show here that the frequencies of Id+ B cells are comparable in both strains, but their state of activity is sharply distinct: while all Id+ B cells are small, resting lymphocytes in C57BL/6 mice, they are all large, naturally activated cells in BALB/c mice. The suggestion that naturally activated cells are poorly engaged in specific responses was supported by the delayed and lower Id+ responses obtained in BALB/c mice when they are immunized, in parallel with C57BL/6 animals, with a conjugate of anti-Id antibodies and lipopolysaccharide. Finally, C57BL/6 responder mice were found to closely reproduce the normal BALB/c situation, if analyzed 3 months after anti-Id priming: they produce low levels of serum Id and all Id+ B cells are in the large lymphocyte compartment. Upon immunization these animals develop serum Id+ responses that are undistinguishable from low-responder BALB/c mice. The relevance of these observations for the questions of physiologic self-reactivity is discussed.

Animals↗
PubMed1988

"Radioresistant" intrathymic T cell precursors express T cell receptor C gamma 4- and C delta-specific gene messages.

We have studied the expression and sequences of T cell receptor gamma and delta chain gene messages in intrathymic T cell precursors of mice irradiated with 600 rads. On day 7 after irradiation a high level of expression of gamma and delta chain messages was detected in thymocytes which were composed of a relatively high proportion of CD3+CD4-CD8- thymocytes. During further development of the precursors from day 7 to day 14 after irradiation, gamma and delta chain messages fell to low levels and alpha and beta mRNA levels increased. Nucleotide sequence analysis of 14 gamma and 10 delta chain complementary DNA (cDNA) in the thymocytes on day 7 revealed that there were 7 functional gamma chain transcripts composed of V gamma 2-J gamma 2-C gamma 2 or V gamma 1-J gamma 4-C gamma 4 gene segments, and only 1 functional delta chain transcript composed of the V delta M23-D delta 1-D delta 2-J delta 1-C delta gene segments. The repertoire of gamma chain and delta chain genes used in "radioresistant" intrathymic T cell precursors of adult mice appears to be limited.

Amino Acid Sequence↗
PubMed1989

Cytotoxic T lymphocyte recognition of HLA-A2 antigens in normal and HLA-Cw3-transgenic mice.

It is well established that a large fraction of murine cytotoxic T cells can recognize allogeneic major histocompatibility complex (MHC) antigens, and that the majority of this response are not restricted by H-2 antigens of the responding host. In contrast, the murine response against the xenogeneic HLA class I antigens is relatively weak. Moreover, a large proportion of the responding murine T cells do not recognize the HLA antigen per se but only in an H-2-restricted manner, probably as an HLA peptide bound to H-2. Considerable evidence suggests that in mice the T cell repertoire is selected by thymic H-2 antigens. Therefore, we asked the question whether in transgenic mice expressing an HLA class I antigen the T cell repertoire would be shaped toward a more effective recognition of other HLA alleles. Normal C57BL/6 (B6) and HLA-Cw3-transgenic B6 mice were immunized with a B6-derived cell line transfected with HLA-A2. The resulting A2-specific CTL were tested on L cells transfected with either A2 alone, which should identify the H-2-unrestricted CTL, and on L cells transfected with A2 plus H-2b genes, which should identify the sum of H-2b-restricted and unrestricted CTL. Both bulk culture and limiting dilution experiments showed that the CTL precursor frequencies for A2-specific CTL were not increased in the transgenic mice, and that both strains produced comparable proportions of H-2b-restricted and of unrestricted A2-specific CTL. The B6.Cw3 mice seemed to respond less well to HLA-A2 than the normal B6 mice, suggesting the possibility of tolerance for peptides shared by the Cw3 and A2 molecules. In conclusion, the T cells in the B6.Cw3 transgenic mice did not seem to be selected towards a stronger and more unrestricted recognition of an allo-HLA antigen. The possible reasons are discussed.

Animals↗
PubMed1989

Combinations of interleukins 2, 4 and 5 regulate the secretion of murine immunoglobulin isotypes.

Immunoglobulin (Ig) production by lipopolysaccharide (LPS)-stimulated cells in the presence of various combinations of interleukin (IL)2, IL4 and IL5 was examined. IgG1, IgM and IgE secretion was studied using a 3T3-fibroblast filler cell-supported B cell culture system, either at low cell density to support maximal Ig secretion, or at limiting dilution to determine isotype-specific precursor frequencies. In the presence of optimal concentrations of IL5 (2%) and IL2 (3 U/ml), the addition of 1 U/ml of IL4 resulted in the production of 4 ng of IgG1 per input B cell. In contrast, 1000 U/ml of IL4 alone was required to produce equivalent levels of IgG1. IL5 and IL2 increased both the precursor frequency and the amount of IgG1 secreted per clone in the presence of low levels of IL4. On the other hand, IgM secretion was decreased 10-fold by the addition of 10 U/ml IL4 or greater. This was not seen when IL5 was present. The IgM-secreting precursor frequency was unaffected by any of the lymphokines, either singly or in combination. The inhibition of IgM production and subsequent relief of this with IL5 was shown to affect the amount of IgM secreted per clone. IgE secretion was shown to be highly IL4 dependent with only minor reduction in the required concentration following addition of IL5 and IL2. At the clonal level, the majority of IgE-secreting clones (93%) at high IL4 concentrations (200 U/ml) arose from precursors which were able to produce IgM and IgG1. Furthermore, only 3% of the clones secreted IgG1 alone, with a further 3% secreting IgE alone. These results suggest that B cells in vivo are predominantly uncommitted in terms of isotype to be produced, the choice of isotype secreted being dependent on the nature of the stimulus. Overall, this work shows that the isotype secreted by B cells can be regulated using combinations of IL2, IL4 and IL5, and that major effects can be achieved by very small quantities of lymphokines acting in synergy.

Animals↗
PubMed1993

Increased number of cytotoxic T cells within CD4+8- T cells in beta 2-microglobulin, major histocompatibility complex class I-deficient mice.

Targeted disruption of beta 2-microglobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4-8+ T cells. Despite this, beta 2 M-/- mice reject skin grafts and cope with most viral infections tested. We asked whether CD4+8- cytotoxic T cells would play a role in compensating for the defect in CD4-8+ cytotoxic T cell function. We found that the cytotoxic activity against class II+ targets is significantly higher among CD4+8- T cells of beta 2M-/- than among those of beta 2M+/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4+8-, class II-specific T cells is at least fivefold higher in beta 2M-/- than in beta 2M+/+ mice. These results suggest that CD4+8- cytotoxic T cells could play a major role in carrying out cytotoxic function in beta 2M-/- mice.

Animals↗
PubMed1995

Lack of effect of chronic morphine treatment and naloxone-precipitated withdrawal on tyrosine hydroxylase, galanin, and neuropeptide Y mRNA levels in the rat locus coeruleus.

Morphine dependence was experimentally induced in rats by daily injection of increasing doses of morphine for seven days. Withdrawal was precipitated in half of the morphine-dependent rats by a single injection of naloxone on day 8. Behavioral signs of withdrawal were evident in the morphine/naloxone group. Gene expression in locus coeruleus (LC) neurons was investigated using quantitative in situ hybridization analysis. Messenger RNA (mRNA) levels for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and for precursors to galanin (GAL) and neuropeptide Y (NPY), peptides that coexist with norepinephrine in LC neurons, were not altered by chronic morphine treatment or naloxone-precipitated withdrawal. In contrast, mRNA levels for c-fos were dramatically elevated in the LC following naloxone-precipitated withdrawal. Chronic morphine treatment caused a small decrease in levels of mRNA encoding the precursor to corticotropin-releasing factor (CRF) in Barrington's nucleus. Although long-term adaptations of LC neurons have previously been implicated in the development of morphine tolerance, dependence, and withdrawal, alterations in the levels of TH, GAL, or NPY mRNA in the LC apparently do not underlie this process.

Animals↗
PubMed2006

Stem cell therapy for Parkinson's disease.

Transplantation of fetal dopaminergic (DA) neurons can produce symptomatic relief for patients with Parkinson's disease, but the technical and ethical difficulties have limited the application of this therapy. Neural precursor cells and embryonic stem cells (ESCs) are expected to be candidates of potential donor cells for transplantation. Human neural precursor cells obtained from the midbrain give rise to TH-positive neurons. The growth of the cells, however, is slow and the differentiation rate of DA neurons is still low for clinical application. Monkey ESCs give rise to midbrain DA neurons, and the transplanted ESC-derived neurospheres function as DA neurons, attenuating the neurological symptoms of the monkey Parkinson's disease model. These results suggest the possibility of using stem cells for the treatment of Parkinson's disease, but problems such as the low survival rate in vivo and tumor formation must be solved.

Animals↗
PubMedDate not supplied

Prophylaxis of venous thromboembolism.

The objective of prophylaxis in venous thromboembolism is, first, to prevent fatal pulmonary embolism and, second, to reduce the morbidity associated with deep vein thrombosis (DVT) and the postphlebitic limb. This should now be standard practice for most patients over 40 years of age undergoing major surgery and for younger patients with a history of venous thromboembolism. Particularly high-risk groups include patients over 60 years of age undergoing major surgery, those with malignancy, and those requiring hip operations. Low-dose subcutaneous heparin 5,000 IU commencing 2 hours preoperatively and continuing 12 hourly until the patient is fully mobile is unequivocally effective in preventing DVT in medical and surgical patients and, most importantly, significantly reduces the incidence of fatal postoperative pulmonary embolism and total mortality. Such prophylaxis, in the presence of established DVT, also limits proximal clot propagation, which is the precursor of major pulmonary embolism. Low-dose heparin prophylaxis is associated with a small risk of bleeding complications, evidenced mostly by an increased frequency of wound hematoma rather than major clinical hemorrhage. Low molecular weight heparin fragments (e.g., Fragmin, Choay, Enoxaparine) are emerging as useful alternative agents, having the advantage of once daily administration and yet providing similar efficacy in the prevention of DVT. Mechanical methods of prevention which counteract venous stasis, such as graduated elastic compression stockings, are also useful in protecting against DVT but have not been shown to prevent fatal postoperative pulmonary embolism. They are recommended particularly for patients in whom heparin prophylaxis is best avoided (e.g., neurosurgery) and possibly in combination with heparin in very high-risk patients.

Anticoagulants↗
PubMed1984

Goitrin--a nitrosatable constitutent of plant foodstuffs.

N-nitrosamides are known as direct-acting carcinogens at the site of their formation; they do not need any metabolic activation in vivo. The conditions leading to their formation in the stomach, and also their genotoxicity, have been thoroughly studied with some model compounds. Several reports link this type of compound to the induction of gastric cancer in human. However, only limited data are presently available about possible precursors of N-nitrosamides in foods. In the present study we found that goitrin --a naturally occurring compound in cruciferous vegetables and rape--could be easily nitrosated by treatment with nitrite under stomach conditions, yielding with loss of sulfur the N-nitroso- oxazolidone 4 (fig.). This product has a mutagenicity pattern and potency similar to that of N-nitroso-N-methyl-N'- nitroguanidine (MNNG) in the Ames Salmonella/mammalian microsome test.

Biotransformation↗
PubMed2006

Expression profile of a PAL gene from Astragalus membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis.

An important traditional Chinese medicine herb, Astragalus membranaceus var. Mongholicus, whose dried root is known as Radix astragali ("Huangqi" in Chinese), has high flavonoid content as an essential active constituent. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the first and also a rate-limiting step in phenylpropanoid pathway, which supplies precursors for a variety of secondary metabolites including flavonoids. A PAL gene, designated AmPAL1 (GenBank accession no. AY986506), was isolated from A. membranaceus var. Mongholicus with a full-length cDNA of 2562 nucleotides and an open reading frame of 2154 bp. Northern blot analysis revealed that AmPAL1 expressed universally in different organs, and its expression was markedly induced by UV irradiation, mechanical wounding, and white light irradiation on etiolated seedlings, with some distinctive responsive properties. Content of a typical flavonoid, quercetin, in A. membranaceus var. Mongholicus of different ages correlated with PAL enzymatic activity. Transgenic tobacco plants harboring AmPAL1 under the control of the CaMV35S promoter showed significantly increased PAL activity and correlatively increased quercetin content than those in non-transformed plants. These results indicate that PAL is maybe a key point for flux into flavonoid biosynthesis in the genetic control of secondary metabolism in A. membranaceus var. Mongholicus.

Astragalus Plant↗
PubMed1999

Immunohistochemical evidence for the presence of tryptophan hydroxylase and serotonin in the rodent Harderian gland.

The Harderian gland is considered as being an extrapineal source of melatonin. In most rodents, the Harderian gland contains two epithelial cell types (I and II). The aim of this study has been to define which cell type is involved in indoleamine synthesis. The presence and localization of serotonin (melatonin precursor) and tryptophan hydroxylase (the rate-limiting enzyme for serotonin synthesis) have been investigated by immunohistochemistry in male Wistar rats, Syrian hamsters and Djungarian hamsters. The results of the present study show that immunoreactivity for tryptophan hydroxylase and serotonin is confined to the type I cell, suggesting that this cell type is involved in indoleamine synthesis in the rodent Harderian gland.

Animals↗
PubMed1989

A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes.

We have developed a modified primer extension procedure for specific detection of mRNA. Alkali-fragmented total cellular RNA or some RNA fraction is hybridized to single-stranded or double-stranded M13 DNA containing the insert of interest which is immobilized on nylon membranes. Hybridized RNA is then detected by incubation of membranes with Escherichia coli RNase H and DNA polymerase I. RNase H is used for nicking the RNA in the hybrids. The resulting 3'-OH groups can subsequently be used by DNA polymerase I to synthesize a labeled complementary strand. The method described is both relatively fast and sensitive and particularly useful for screening large numbers of DNA clones for their representation in RNA populations. Using total cellular RNA as hybridization probe and single-stranded M13 DNA as template as low as 0.25 ng of a specific mRNA was detected (2.5-fold background) when adding 1 microCi [3H]dCTP or 2.5 microCi [32P]d-CTP alternatively as radioactive precursor for the labeling reaction. The detection limit increased to 1 ng (2-fold background) with denatured replicative form double-stranded M13 DNA as template.

Animals↗
PubMed1980

The contribution of serum phosphatidylcholine and lysophosphatidylcholine to lymph phosphatidylcholine.

1. The contribution of serum phosphatidylcholine and 1-acyl lysophosphatidylcholine to chylomicron and mesenteric lymph lipoproteins of heavier buoyant density was studied in rats with catheters placed in the jugular vein, duodenum, common bile duct and mesenteric lymph duct. The effect of including 10 mM phosphatidylcholine in the triolein emulsion infused into the duodenum was also studied. 2. The intravenous infusion of phosphatidylcholine did not affect delivery of phosphatidylcholine into the lymph when phosphatidylcholine was included in the duodenal infusion. However, on intravenous lysophosphatidylcholine infusion, phosphatidylcholine transport into the lymph was increased both in chylomicrons and the other lipoproteins found in the lymph when phosphatidylcholine was included in the duodenal infusion. 3. The incorporation of serum phosphatidylcholine into chylomicron phosphatidylcholine was minimal and decreased further by intraduodenal phosphatidylcholine infusion. Incorporation into the heavier lymph lipoproteins was less than 20%. 4. The incorporation of serum lysophosphatidylcholine into chylomicrons was 17% at 4--6 h of infusion which was decreased by intraduodenal phosphatidylcholine as was the incorporation into the heavier lipoproteins of lymph. 5. It is concluded that serum phosphatidylcholine is a poor precursor of chylomicron phosphatidylcholine and that while lysophosphatidylcholine is a somewhat better precursor, its contribution to chylomicron phosphatidylcholine is limited by its serum concentration.

Animals↗
PubMed1987

Putrescine derivatives as substrates of spermidine synthase.

1. Derivatives of 1,4-butanediamine (putrescine) were studied in vitro and in vivo as potential substrates of spermidine synthase. 2. Substituents in the 1-position decreased the reaction rate by steric hindrance, and in the case of electron withdrawing groups there was an additional decrease due to the lowered basicity of the vicinal amino group. 3. Substituents in the 2-position are tolerated; under saturating conditions reaction rates are comparable to those of putrescine. 4. Compounds which were identified as substrates of spermidine synthase in vitro formed derivatives of spermidine and spermine in vivo. Exception: compounds, such as 1-methylputrescine formed in vivo only a spermidine derivative, because the second aminopropylation was sterically hindered by the substituent on the carbon atom next to the amino group. 5. Administration of 2-hydroxyputrescine to alpha-difluoromethylornithine-pretreated chick embryos produced spermidine and spermine analogues in amounts exceeding spermidine and spermine formation from putrescine under comparable conditions. 6. Since the concentration of 2-hydroxyputrescine in the embryo was higher than that of putrescine and all other putrescine analogues, it appears that uptake of the polyamine precursor from the yolk may be rate limiting. 7. Three days after administration of 5 mM alpha-difluoromethylornithine there is a near-to-complete arrest of embryonal growth. 8. A series of diamines supported growth under these conditions, even if they were not substrates of spermidine synthase. 9. Survival of chick embryos was, however, only supported if the diamines were capable of forming significant amounts of spermidine and spermine analogues.

Animals↗
PubMed1992

Non-cholesterol sterols, absorption and synthesis of cholesterol and apolipoprotein A-I kinetics in a Finnish lecithin-cholesterol acyltransferase deficient family.

We describe the first Finnish LCAT-deficient family with two affected, one questionably affected and one healthy family member. The affected family members presented stomatocytes in the peripheral blood, exhibited low serum levels of total, LDL and HDL cholesterol, triglycerides, phospholipids and apolipoprotein A-I and especially A-II. Apolipoprotein A-I catabolism was accelerated to moderately high and very high levels in the two affected subjects. Cholesterol esterification percentage was low in all lipoprotein fractions. The intestinal cholesterol absorption efficiency and cholesterol and bile acid synthesis were within normal limits. The esterification percentage of demethylated cholesterol precursor sterols, cholestanol and plant sterols resembled mostly that of cholesterol, while those of VLDL and LDL methostenols, precursor sterols esterified by acyl-CoA:cholesterol acyltransferase (ACAT), suggested normal ACAT activity. In HDL all sterols were poorly esterified. The observations on stomatocytes, normal absorption and synthesis of cholesterol and bile acids, abnormal kinetics of apolipoprotein A-I, evidence of normal ACAT activity and abnormal esterification of non-cholesterol sterols are findings presented for the first time in LCAT deficiency.

Absorption↗
PubMed1981

A new assay for cytotoxic lymphocytes, based on a radioautographic readout of 111 In release, suitable for rapid, semi-automated assessment of limit-dilution cultures.

A new assay for cytotoxic T lymphocytes is described, of general application, but particularly suitable for rapid, semi-automated assessment of multiple microculture tests. Target cells are labelled with high efficiency and to high specific activity with the oxine chelate of 111 indium. After a 3-4 h incubation of test cells with 5 X 10(3) labelled target cells in V wells of microtitre trays, samples of the supernatant are spotted on paper (5 microliter) or transferred to soft-plastic U wells (25-50 microliter) and the 111 In release assessed by radio-autography. Overnight exposure of X-ray film with intensifying screens at -70 degrees C gives and image which is an intense dark spot for maximum release, a barely visible darkening with the low spontaneous release, and a definite positive with 10% specific lysis. The degree of film darkening, which can be quantitated by microdensitometry, shows a linear relationship with cytotoxic T lymphocyte dose up to the 40% lysis level. The labelling intensity and sensitivity can be adjusted over a wide range, allowing a single batch of the short half-life isotope to serve for 2 weeks. The 96 assays from a single tray are developed simultaneously on a single small sheet of film. Many trays can be processed together, and handling is rapid if 96-channel automatic pipettors are used. The method allows rapid visual scanning for positive and negative limit dilution cultures in cytotoxic T cell precursor frequency and specificity studies. In addition, in conjunction with an automated densitometer designed to scan microtitre trays, the method provides an efficient alternative to isotope counting in routine cytotoxic assays.

Animals↗
PubMed1987

Eicosadiynoic acid: a non-toxic inhibitor of multiple enzymatic steps in the production of icosanoids from arachidonic acid.

We have studied the acetylenic fatty acid 20:2 delta 8a, 11a (eicosadiynoic acid, EDYA). It was found that this compound acts as an inhibitor of several steps in the production of icosanoids from arachidonic acid. First, the compound was shown to inhibit arachidonate uptake by platelets. Second, using a detergent solubilized preparation from calf brain, EDYA was found to inhibit both the arachidonoyl and the non-specific long chain acyl-CoA synthetase, which convert arachidonate to its CoA ester. Third, the compound decreased the conversion of dihomo gamma linolenic acid to arachidonate in the mouse fibrosarcoma HSDM1C1 cell line, acting as an apparent delta 5 desaturase inhibitor. Finally, EDYA (50 microM) inhibited cyclooxygenase activity. The compound was not toxic to cultured cells. Cells were grown for months in tissue culture medium at concentrations as high as 50 microM, with no morphologic changes by light microscopy and no prolongation of the doubling time over untreated cells. Our findings with this compound indicate that it limits icosanoid production by inhibiting cyclooxygenase and also by limiting arachidonate uptake, activation, and production from precursor fatty acids.

Animals↗