Intracellular and cell surface mapping of HLA DR in the presence and absence of the invariant chain.
Explore the source record for details and available documents.
Cell Line↗
Biomedical subjects
N Fernandez
Publications and source records attributed to N Fernandez.
The state of aggregation of MHC class II molecules at the cell-surface is temperature dependent.
Explore the source record for details and available documents.
Cell Line↗
Single particle imaging of cell-surface HLA-DR tetramers.
Explore the source record for details and available documents.
Crystallography, X-Ray↗
Bovine somatotropin dose titration in lactating dairy ewes. 2. Dose determination and factors affecting the response.
Seventy-four lactating dairy ewes were injected with recombinant bovine somatotropin (bst; sometribove) in a sustained-release formulation. Ewes received 0, 80, 160, or 240 mg of bST/14 d from wk 3 to 8 of lactation (part 1) and 0, 80, or 160 mg of bST/14 d from wk 11 to 18 of lactation (part 2). The optimal dose of bST was studied as well as the factors (lactation stage, lactation number, initial milk production, body weight, and body condition) possibly affecting the increase in milk production following bST injection. Using a quadratic regression model, the maximum theoretical dose was determined to be 181 mg of bST/14 d during the first part of lactation. During the second part of lactation, 143 mg of bST/14 d was the maximum theoretical dose. The increment of milk production did not vary with lactation number, but first lactation ewes, in general, responded better than did multiparous ewes. Relative to initial milk production, improvement was greatest for ewes with average milk production (1500 ml/d) that received a dose of 192.3 mg of bST/14 d during the first part of lactation; improvement was also measured from the highest producers (2000 ml/d) during the second part of lactation. The best response was obtained from ewes with average body condition (score 3 on a five-point scale where 1 = thin to 5 = fat) and a dose of 200 mg of bST/14 d during the first part of lactation; during the second part of lactation, body condition score had no effect. Body weight had no effect on the increment of production at any time.
Animal Nutritional Physiological Phenomena↗
Organochlorine pesticide residues in bovine milk from Leon (Spain).
Residue levels of the organochlorine pesticides (alpha-HCH, lindane, heptachlor-epoxide, aldrin, endrin, dieldrin, o,p'-TDE, p,p'-TDE, p,p'-DDE and p,p'-DDT) were determined in raw bovine milk and compared with the maximum levels allowed by the European Union (EU) in these foods. The highest incidence percentage of the ten insecticides measured was for lindane, followed by alpha-HCH and aldrin. Moreover, the highest mean residue level was for alpha-HCH. None of the samples analyzed exceeded the maximum levels allowed by the EU.
Animals↗
Strategies for cancer gene therapy using adenoviral vectors.
Modification of tumor cells using gene transfer either to enhance host immunity or to act directly on tumor cells is being intensively studied in animal models. Remarkable results have yielded to approved clinical protocols in the treatment of cancer patients using this approach. Several methods of gene delivery have been developed. This article is particularly devoted to the interest of the use of adenoviral vectors in the different strategies of cancer gene therapy.
Adenoviridae↗
A gradient of cytoplasmic Cactus degradation establishes the nuclear localization gradient of the dorsal morphogen in Drosophila.
Dorsoventral axis formation in the Drosophila embryo is established by a signal transduction pathway that comprises the products of at least 12 maternal genes. Two of these genes, dorsal and cactus, show homology to the mammalian transcription factor NF-kappa B and its inhibitor I kappa B, respectively. As in the case for I kappa B and NF-kappa B, Cactus inhibits Dorsal by retaining it in the cytoplasm. In response to the signal produced and transmitted by the products of the other genes, Dorsal translocates to the nucleus preferentially on the ventral side of the embryo. Here, we show that Cactus forms a cytoplasmic concentration gradient inversely correlated to the nuclear translocation gradient of Dorsal. Deletions of the N-terminus and C-terminus of Cactus reveal that two modes of degradation control cactus activity: signal-induced degradation and signal-independent degradation, respectively. Genetic evidence indicates that degradation of Cactus is required, but not sufficient to translocates Dorsal completely into the nucleus.
Animals↗
Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging.
The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were obtained by sequential imaging over a period of typically 30 minutes. Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200-800 nm. Diffusion coefficients for random diffusion were in the range 1 x 10(-13)-5 x 10(-12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.
Antibodies, Monoclonal↗
[Interferon alfa in the treatment of chronic hepatitis C: course of 24 versus 48 weeks. Results of a randomized trial].
UNLABELLED: The aim of this trial was to investigate if a more prolonged course of interferon (IFN) is able to increase the long-term benefit in patients with chronic hepatitis C. Forty-four patients with active chronic hepatitis and antibodies to HCV were randomly assigned to receive IFN-alfa 2b 3 MU t.i.w. during 24 weeks (group I, n 23) or during 48 weeks (group II, n 21). In the evaluation of results, complete response was considered when the ALT values returned to normality during the treatment; and sustained response, when the ALT values persisted below normal range during at least 6 months post therapy. Histologic changes were compared by using the Histological Activity Index, or Knodell score. Viremia status was evaluated for the study of HCV RNA (by nested-RT-PCR). RESULTS: There were no significant differences between both groups before treatment, in terms of age, sex, ALT, or histologic findings (11 patients in group I, and 7 in group II had cirrhosis). Complete response was found in 9 patients (39.1%) from group I; in 11 (52.4%) from group II (NS). Basal histologic findings were identified as the only predictive factor of complete and sustained response, by logistic regression analysis. Considering only noncirrhotic patients, complete response was seen in 58.3% in patients from group I, 71.4% in group II. Sustained response was obtained in 4 patients from group I, (17.4%), 7 from group II (33.3%) (NS). Post IFN liver biopsies were performed in 23 patients (12 from group I, 11 from group II). In group I patients, there were no significant changes. In group II, Knodell score was found to be significantly decreased post IFN [pre IFN, median 10, range 3-15; post IFN, median 6, range 2-14] (p < 0.05). HCV RNA was absent in serum during the follow-up post IFN in 2 patients from group I, in 3 from group II. The results of this study show that a 48 weeks course of IFN has a trend to achieve a higher sustained response than the usual regime (but non significant); and it produces a decrease in the histologic activity. The best predictive factor of positive response was the absence of cirrhosis in our study (although we did not evaluate viral factors, such as genotypes or HCV viremia levels).
Adult↗
Endothelin-1-induced in vitro cerebral venoconstriction is mediated by endothelin ETA receptors.
The in vitro effects of endothelin-1 on cerebral veins were studied using cylindrical segments, 5 mm long, from dog pial veins. Isometric responses to endothelin-1 (10(-12)-10(-7) M) and to the endothelin ET(B) receptor agonist, IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21), 10(-12) -10(-7) M), were recorded in veins under control conditions and pretreated with the endothelin ET(A) receptor antagonist, BQ-123 (cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp), 10(-8) -10(-5) M), and the endothelin ETB receptor antagonist, BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(me thoxycarbonyl)-D-tryptophyl]-D-norleucine monosodium, 10(-6) and 10(-5) M). The response to endothelin-1 was also recorded in veins pretreated with the nitric oxide synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), or the cyclooxygenase inhibitor, meclofenamate (10(-5) M), and in veins without endothelium or placed in medium without Ca2+ but with EDTA (0.1 mM). In control veins, endothelin-1 produced a concentration-dependent contraction (EC50 = 2.0 x 10(-10) M; maximal contraction = 113 +/- 6 mg) and IRL 1620 induced no effects or a small contraction only with high concentrations (10(-8) - 10(-6) M) (EC50 = 1.5 x 10 (-8) M; maximal contraction = 9 +/- 3 mg). BQ-123 shifted the response to endothelin-1 to the right in a parallel, concentration-dependent way, whereas BQ-788, L-NAME or meclofenamate did not modify the response to endothelin-1. Compared with the control, veins in a medium without Ca2+ had similar EC50 values, but a lower maximal contraction induced by endothelin-1 (57 +/- 10 mg, P < 0.05), and veins without endothelium exhibited similar EC50 values. Thus, endothelin-1 produces marked cerebral venoconstriction that could be mainly mediated by activation of endothelin ETA receptors, may be dependent on extracellular Ca2+, and may be independent of endothelium, nitric oxide and prostanoids.
Animals↗
Cerebral reactive hyperaemia and arterial pressure in anaesthetized goats.
The effects of arterial pressure on cerebral reactive hyperaemia were studied in anaesthetized goats measuring electromagnetically middle cerebral artery flow and performing arterial occlusions of 5-30 s. Under normotension (mean arterial pressure, MAP = 11 +/- 0.3 kPa), reactive hyperaemia (peak hyperaemic flow to control flow and repayment to debt ratios) increased, and cerebrovascular resistance during peak hyperaemic flow decreased, as ischaemia duration lengthened; the virtual maximal changes were obtained after 20 s ischaemia. During hypertension by aorta constriction (MAP = 18 +/- 0.7 kPa) or by i.v. infusion of noradrenaline (MAP = 19 +/- 0.8 kPa) middle cerebral artery flow did not change significantly and cerebrovascular resistance increased 25 and 46%, respectively (P < 0.05). During both types of hypertension reactive hyperaemia was over 50% higher, and the decrement in cerebrovascular resistance during peak hyperaemic flow was also higher, than under normotension. During hypotension by constriction of the inferior vena cava (MAP = 5 +/- 0.5 kPa) or by i.v. infusion of isoproterenol (MAP = 6 +/- 0.5 kPa), middle cerebral artery flow decreased 35% or did not change, and cerebrovascular resistance decreased 41 and 45%, respectively (P < 0.05). In these conditions, reactive hyperaemia and the decrement in cerebrovascular resistance during peak hyperaemic flow were reduced 80%, and it was similar in both types of hypotension. The absolute levels of cerebrovascular resistance obtained during peak hyperaemia were similar during normotension, hypertension and hypotension. Thus, arterial pressure is a main determinant of postocclusive cerebral reactive hyperaemia, and myogenic mechanisms may be of significance in determining the early stage of cerebral reactive hyperaemia after brief ischaemias. Adrenergic mechanisms might be of minor significance in this type of cerebral reactive hyperaemia.
Anesthesia↗
Bovine somatotropin dose titration in lactating dairy ewes. 1. Milk yield and milk composition.
Seventy-four lactating dairy ewes were injected with recombinant bST (sometribove) in a sustained-release formulation. Ewes received 0, 80, 160, or 240 mg of bST every 14 d from wk 3 to 8 of lactation (part 1) and 0, 80, or 160 mg of bST every 14 d from wk 11 to 23 of lactation (part 2). Sometribove increased milk yield over that of the controls for all treatment groups. The increase was largest for the group that was administered 160 mg of bST: milk yield was 34.1 and 53.2% and 6% FCM was 36.9 and 51.8% for parts 1 and 2 of the study, respectively. Sometribove increased milk fat during part 1 of the study, but decreased milk fat during part 2. Protein contents of milk were decreased throughout the study. For all group, bST increased the yield of milk constituents over that of the controls. When milking frequency was reduced from twice to once daily, the difference in milk yield between control ewes and those treated with bST was maintained. Neither mastitis incidence nor milk SCC were affected by bST treatment. Recombinant bST is efficacious in increasing both actual milk yield and 6% FCM over the dose range of 80 to 240 mg/14 d without adverse effects for lactating ewes.
Animals↗
[Application of gene therapy to antitumoral vaccination].
Explore the source record for details and available documents.
Animals↗
Analysis of mRNA for class I HLA on human gametogenic cells.
We have studied mRNA expression for Class I HLA (human leukocyte antigen) on male germ cells by amplification of gene fragments in PCR technique and by Northern hybridization. RNA was extracted from fractionated gametogenic cells (isolated from testis) and reversely transcribed. Then, cDNA was amplified for specific HLA sequence (HLA, -A, -B, -C). The specificity of this product was confirmed in "nested" PCR of 400 bp gene fragment coding for alpha 2 domain, alpha 3 domain, and the transmembrane portion of Class I HLA. The results indicate minimal expression of classical Class I HLA on gametogenic cells. Northern hybridization with 669 bp cDNA fragment (spanning for alpha 3 domain, transmembrane, cytoplasmic, and 3' untranslated region) resulted in a low intensity signal from gametogenic cell fractions and confirmed our findings obtained by PCR. The minimal expression of classical HLA antigens may create a neutral cover for the male reproductive system, thereby preventing an immunological response during germ cell differentiation.
Blotting, Northern↗
Release from a human monocyte-like cell line of two different soluble forms of the lipopolysaccharide receptor, CD14.
Lipopolysaccharide (LPS) stimulates mononuclear phagocytes to synthesize and secrete immunoregulatory and inflammatory molecules such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha). LPS forms complexes with either the serum protein termed LPS-binding protein or a serum factor, septin. These complexes are more stimulatory than LPS alone. The myeloid differentiation antigen CD14 is known to be the receptor for such complexes. In the present study, by using a monocytic cell line, we demonstrate the release of two different soluble forms of CD14 (sCD14) which are secreted by different mechanisms. We show that the two sCD14 forms differ in their electrophoretic mobility, two-dimensional gel electrophoretic patterns, sensitivity to endoglycosidases and peptide maps. One of the sCD14 molecules, apparent molecular mass 48 kDa, was found in supernatants of both surface iodinated and [35S]methionine biosynthetically labeled cells. The other sCD14 molecule (56 kDa) was found labeled only in supernatants of [35S]methionine-labeled cells. Furthermore, purified 48 kDa sCD14 enhanced the LPS-induced TNF-alpha and IL-6 release by the monocytic cells suggesting that a cell-surface signal transducer molecule may be involved in signaling. The data suggest a possible novel role for sCD14 in the monocyte response to LPS.
Antigens, CD↗
Preimplantation mouse embryos express Mhc class I genes before the first cleavage division.
We have used oligonucleotide primers complementary for polymorphic regions of the mouse H-2D gene in a highly sensitive polymerase chain reaction (PCR) assay to detect the transcription of maternal and paternal class I mRNAs in gametes and preimplantation embryos. Using congenic strains of mice differing only at the major histocompatibility loci, class I (H-2D) mRNA of both the maternal and paternal haplotypes was demonstrated in embryos from the one-cell zygote to the late blastocyst stage of development but could not be detected in vas deferens or in vitro capacitated sperm or in ovulated secondary oocytes. These data clearly show that both paternally and maternally inherited Major histocompatibility complex (Mhc) class I genes are transcribed from the earliest stages of embryonic development, and suggest that developmental regulation of expression of their protein products is principally at the post-transcriptional level.
Animals↗
Unusual expression pattern of the MHC class I Q5k gene in the AKR mouse: possible role in embryo development.
Analysis of the tissue-specific expression of the Q5k gene in the AKR mouse reveals an unusual expression pattern. The Q5k mRNA is present in embryos from day 12, but expression is switched off in most tissues except thymus and testis shortly after birth. Late in pregnancy the gene is again transcribed in females. Analysis at the epitope level, with a Qa-2 specific monoclonal antibody revealed that in most cases the Q5k product is confined to the cytoplasm. These results suggest that Q5k has a most unusual tissue distribution and timing of expression among all the H-2 class I and Q genes so far described.
Animals↗
CD14 and tolerance to lipopolysaccharide: biochemical and functional analysis.
To evaluate the role of the high-affinity monocyte receptor for lipopolysaccharide (LPS), CD14, in the process of tolerance to LPS, the human monocytic cell line Mono-Mac-6 was cultured in the absence or presence of different amounts of LPS. The kinetics of CD14 modulation in these cells showed an initial 4-day period characterized by increased cell-surface expression, rate of biosynthesis (peaking at 48 hr) and release of its soluble forms (sCD14) which correlated with the amount of LPS in the culture. At this time, tolerance to LPS was already established, as measured by tumour necrosis factor-alpha (TNF-alpha) induction, it was LPS dose dependent and persisted up to 15 days. LPS also reduced the cell proliferation rate in a dose-dependent manner. After 8 days and up to 15 days, the CD14 biosynthesis, cell-surface expression and release of sCD14 inversely correlated with the level of LPS in the culture. The 48-hr LPS-pretreated cells showed a slightly decreased CD14 affinity for LPS, a relative high number of CD14 molecules per cells, and desensitization also to a phorbol 12-myristate 13-acetate (PMA) challenge. An anti-CD14 monoclonal antibody (mAb) protected the cells from tolerization when added at the beginning of culture, as revealed by challenge with LPS and PMA. The data indicate that in this model tolerization to LPS (1) precedes CD14 down-modulation, (2) operates by alteration of the receptor affinity for LPS and by a mechanism which affects a protein kinase C (PKC)-dependent signalling pathway, and (3) that CD14 plays a critical role in the establishment of tolerance to LPS. In addition, analysis of the data suggests the existence of a PKC-independent signalling pathway for LPS tolerization and a CD14-independent mechanism for establishing tolerance.
Antigens, CD↗