Search

Several fusion genes such as BCR::ABL1, FIP1L1::PDGFRA, and PML::RARAare now efficiently targeted by specific therapies in patients with leukemia. Although these therapies have significantly improved patient outcomes, leukemia relapse and progression remain clinical concerns. Most myeloid next-generation sequencing (NGS) panels do not detect or quantify these fusions. It therefore remains difficult to decipher the clonal architecture and dynamics of myeloid malignancy patients, although these factors can affect clinical decisions and provide pathophysiologic insights. An asymmetric capture sequencing strategy (aCAP-Seq) and a bioinformatics algorithm (HmnFusion) were developed to detect and quantify MBCR::ABL1, μBCR::ABL1, PML::RARA, and FIP1L1::PDGFRAfusion genes in an NGS panel targeting 41 genes. One-hundred nineteen DNA samples derived from 106 patients were analyzed by conventional methods at diagnosis or on follow-up and were sequenced with this NGS myeloid panel. The specificity and sensitivity of fusion detection by aCAP-Seq were 100% and 98.1%, respectively, with a limit of detection estimated at 0.1%. Fusion quantifications were linear from 0.1% to 50%. Breakpoint locations and sequences identified by NGS were concordant with results obtained by Sanger sequencing. Finally, this new sensitive and cost-efficient NGS method allowed integrated analysis of resistant chronic myeloid leukemia patients and thus will be of interest to elucidate the mutational landscape and clonal architecture of myeloid malignancies driven by these fusion genes at diagnosis, relapse, or progression.

La rinosinusitis aguda (RSA) está causada por la agresión vírica, bacteriana o fúngica de la mucosa rinosinusal. Se define como un proceso inflamatorio en el seno de la mucosa de la cavidad nasal y de uno o varios senos durante un período inferior a cuatro semanas. Es muy frecuente en la población (estimación de una afectación anual de 1 de cada 8 adultos en Estados Unidos, es decir, 3 millones de consultas anuales) y representa uno de los primeros motivos de consulta en medicina general. Se estima que menos del 2% de las RSA son secundarias a una agresión bacteriana y sólo estas RSA deberían recibir un tratamiento antibiótico. Muchos estudios han intentado determinar los criterios diagnósticos y/o radiológicos que pueden orientar al médico hacia la etiología bacteriana de la RSA para evitar la prescripción inútil de un tratamiento antibiótico. La sobreprescripción de antibióticos junto con el aumento de las resistencias bacterianas ha llevado a muchas sociedades científicas a editar en los últimos 10 años recomendaciones para el diagnóstico y el tratamiento de las RSA. Estas recomendaciones se dirigen sobre todo a determinar el lugar de la antibioticoterapia en el tratamiento de las RSA. Este artículo está destinado a una población de otorrinolaringólogos y tiene como objetivo exponer estas principales recomendaciones, pero también describir las situaciones de consulta con el otorrinolaringólogo que son las situaciones de complicaciones de la RSA, pero también los casos de repetición de la RSA después de un intervalo libre sin síntomas.

Diamond-based microelectromechanical systems (MEMS) enable direct coupling between the quantum states of nitrogen-vacancy (NV) centers and the phonon modes of a mechanical resonator. One example, a diamond high-overtone bulk acoustic resonator (HBAR), features an integrated piezoelectric transducer and supports high-quality factor resonance modes into the gigahertz frequency range. The acoustic modes allow mechanical manipulation of deeply embedded NV centers with long spin and orbital coherence times. Unfortunately, the spin-phonon coupling rate is limited by the large resonator size, >100 μm, and thus strongly coupled NV electron–phonon interactions remain out of reach in current diamond BAR devices. Here, we report the design and fabrication of a semiconfocal HBAR (SCHBAR) device on diamond (silicon carbide) with f× Q> 1012(>1013). The semiconfocal geometry confines the phonon mode laterally below 10 μm. This drastic reduction in modal volume enhances defect center coupling to a mechanical mode by 1000 times compared to prior HBAR devices. For the native NV centers inside the diamond device, we demonstrate mechanically driven spin transitions and show a high strain-driving efficiency with a Rabi frequency of (2π)2.19(14) MHz/Vp, which is comparable to a typical microwave antenna at the same microwave power, making SCHBAR a power-efficient device useful for fast spin control, dressed state coherence protection, and quantum circuit integration.

La rinorrea crónica, tanto anterior como posterior, es un motivo de consulta frecuente. Raras veces aparece de forma aislada y los otros síntomas que la acompañan pueden ser rinológicos, sinusales e incluso extrasinusales. El carácter crónico se define por una duración de más de 12 semanas, de forma continua o intermitente. La hipersecreción de moco es la causa de la rinorrea. Esta hipersecreción se debe a una inflamación o agresión nasal y/o sinusal. Hay muchos mediadores inflamatorios que estimulan esta hipersecreción y suelen activarse o secretarse después de una agresión nasal por irritantes inhalados, alérgenos o ciertos agentes patógenos (virus, bacterias, hongos). La anamnesis permitirá caracterizar la rinorrea, que puede ser uni o bilateral, serosa, mucopurulenta, costrosa o serosanguinolenta, pero también precisar los síntomas asociados. La endoscopia nasal, realizada por un otorrinolaringólogo, es la exploración física clave en caso de duda diagnóstica. Cualquier rinorrea unilateral obliga a sospechar en primer lugar una patología tumoral, sobre todo si existen síntomas asociados (dolor sinusal, hipoestesia del trigémino, trastorno de la motricidad ocular o disminución de la agudeza visual) y requiere una nasofibroendoscopia y una tomografía computarizada del macizo facial.

Many photonic quantum information processing applications would benefit from a high brightness, fiber-coupled source of triggered single photons. Here, we present a fiber-coupled photonic-crystal waveguide (PCWG) single-photon source relying on evanescent coupling of the light field from a tapered outcoupler to an optical fiber. A two-step approach is taken where the performance of the tapered outcoupler is recorded first on an independent device containing an on-chip reflector. Reflection measurements establish that the chip-to-fiber coupling efficiency exceeds 80%. The detailed characterization of a high-efficiency PCWG extended with a tapered outcoupling section is then performed. The corresponding overall single-photon source efficiency is 10.9%±2.3%, which quantifies the success probability to prepare an exciton in the quantum dot, couple it out as a photon in the waveguide, and subsequently transfer it to the fiber. The applied outcoupling method is robust, stable over time, and broadband over several tens of nanometers, which makes it a highly promising pathway to increase the efficiency and reliability of planar chip-based single-photon sources.

A new formulation of magnetostatics is given: it uses the magnetic field h as variable and a penalty technique. For its discretization, a finite element method inside the magnetic materials is combined with a boundary integral method which describes the exterior domain. Numerical tests are presented. The value to be chosen for the penalty parameter and a criterion of validity of the computation are given.

Liver biopsy is considered the gold-standard method for the assessment of liver fibrosis during follow-up of hepatitis C virus-infected patients, but this invasive procedure is not devoid of complications. The aim of the present study was to identify novel non-invasive markers of fibrosis progression. By microarray analysis, we compared transcript levels in two extreme stages of fibrosis from 16 patients. Informative transcripts were validated by real-time PCR and used for the assessment of fibrosis in 23 additional patients. Sixteen transcripts were found to be dysregulated during the fibrogenesis process. Among them, some were of great interest because their corresponding proteins could be serologically measured. Thus, the protein levels of inter-α inhibitor H1, serpin peptidase inhibitor clade F member 2, and transthyretin were all significantly different according to the four Metavir stages of fibrosis. In conclusion, we report here that dysregulation, at both the transcriptional and protein levels, exists during the fibrogenesis process. Our description of three novel serum markers and their potential use as serological tests for the non-invasive diagnosis of liver fibrosis open new opportunities for better follow-up of hepatitis C virus-infected patients.

Les cancers bronchiques à petites cellules (CPC) représentent moins de 20 % de l’ensemble des cancers bronchiques. À cause du risque de dissémination élevé et précoce, seul un tiers est diagnostiqué à un stade localisé. Le traitement des CPC localisés repose sur une association radio-chimiothérapie avec une polychimiothérapie à base de sels de platine et étoposide et une irradiation thoracique de plus en plus souvent conformationnelle. Le pourcentage élevé de rechutes cérébrales a conduit à proposer une irradiation prophylactique cérébrale (IPC) chez les bons répondeurs qui fait donc partie maintenant de la prise en charge standard des patients. Ainsi, dans les formes limitées, les taux de survie peuvent atteindre 25 % à 5 ans mais les rechutes sont fréquentes, même chez des bons répondeurs. Les progrès sont surtout liés à une meilleure intégration de la radiothérapie thoracique et cérébrale avec la polychimiothérapie. Il reste encore de nombreuses questions et cela devrait stimuler plus d’essais thérapeutiques afin d’optimiser les modalités de polychimiothérapie et de radiothérapie (dose, fractionnement optimal), la meilleure façon d’associer ces 2 traitements ainsi que l’éventuelle place de thérapeutiques ciblées.

Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatonin-receptor subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1AmRNA was further confirmed in the PMH by ISH with a 35S-labeled ovine MTNR1Ariboprobe. We next investigated the variation in the expression of MTNR1AmRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1Atranscript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1AmRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1AmRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1Atranscripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT.

Partial hepatectomy (PH)-induced Kupffer cell (KC) activation results in a rapid release of cytokines inducing the acute-phase response (APR). This study was done to evaluate the role of Kupffer cells (KCs) in the course of the APR following PH and a consecutive endotoxin challenge. KC depletion was performed in rats by i.v. administration of 1 mL liposome-encapsulated dichloromethylene diphosohonate (Cl2MDP). Control rats received 1 mL NaCl 0.9%. Forty-eight hours later, PH was performed. At 24 h after PH, rats were randomized to receive either 1mL NaCl 0.9% (saline) or 50 μg/kg LPS i.v. in 1 mL. Animals were sacrificed at 4 h after LPS or saline infusion. The APR was determined by measuring hepatic gene expression of α2-macroglobulin, α1-acid glycoprotein, and IL-6 and expression of hepatic albumin. The APR was significantly depressed in KC-depleted rats. Despite increased IL-6 mRNA synthesis in response to low-dose LPS, no enhancement of acute-phase protein synthesis (APP) was found in KC-depleted rats. Hepatic failure was most profound in KC-depleted rats, as indicated by elevated plasma levels of liver transaminases and ammonia. We conclude that after PH, KC function in the remnant liver is important for the acute-phase reaction and reduces endotoxin-induced hepatocyte damage.

We present two new mixed finite element methods coupled with a boundary method for the three dimensional magnetostatic problem. Such formulations are obtained by coupling a finite element method inside a bounded domain with a boundary integral method involving either the Calderon equations or the inverse of Dirichlet Neumann operator to treat the exterior domain. First, we present the formulations and then prove that our mixed formulations are well posed and that they lead to a convergent Galerkin method. Finally, we give numerical results for a sphere immersed in a homogeneous (source) field in the two formulations. © 2003 Wiley Periodicals, Inc. Numer Methods Partial Differential Eq 19: 443–462, 2003

Genetic variability in plasma melatonin concentrations in ewes results from variations in pineal weight. This study investigated whether it is due to a difference in the number of pinealocytes, or in their size. Two groups of lambs were assigned before birth to being extremes (18 High and 21 Low) by calculating their genetic value on the basis of the melatonin concentrations of their parents. Lambs were bled from 1 week of age until 14 weeks of age. Pineal gland, brain and pituitary weights, length and width of the brain, and length of the hypothalamus were recorded. A significant effect (ANOVA) of genetic group (P<0.05) and age (P<0.05) was detected on mean nocturnal plasma melatonin concentrations, as soon as the first week after birth (mean +/- s.e.m.; High: 51.7 +/- 10.7 vs Low: 31.9 +/- 3.2 pg/ml). There was no difference between the two genetic groups in any of the brain parameters measured, but the pineal glands of the High group were heavier and contained significantly more pinealocytes (High: 27.8 +/- 2.4 vs Low: 21.0 +/- 2.4 x 10(6); P<0.05) than those in the Low group. The mean size of pinealocytes did not differ between the two genetic groups. Thus, the genetic variability in nocturnal plasma melatonin concentrations in sheep is expressed by 1 week of age and higher levels of secretion are the consequence of larger pineal glands containing a greater number of pinealocytes.

We propose mixed and hybrid formulations for the three‐dimensional magnetostatic problem. Such formulations are obtained by coupling finite element method inside the magnetic materials with a boundary element method. We present a formulation where the magnetic field is the state variable and the boundary approach uses a scalar Dirichlet‐Neumann map to describe the exterior domain. Also, we propose a second formulation where the magnetic induction is the state variable and a vectorial Dirichlet‐Neumann map is used to describe the outer field. Numerical discretizations with “edge” and “face” elements are proposed, and for each discrete problem we study an “inf‐sup” condition. © 2002 John Wiley & Sons, Inc. Numer Methods Partial Differential Eq 18: 85–104, 2002

A lipopolysaccharide (LPS)-induced inflammation prior to an hepatic resection has been shown to enhance liver regeneration in rat. The aim of the present study was to investigate the expression of hepatocyte growth factor (HGF) and its c-Met receptor under such experimental conditions. Animals were submitted to a two-third hepatectomy or a LPS challenge carried out 12 h prior to resection. Non parenchymal and parenchymal cells were isolated from livers obtained at various times post-hepatectomy. Quantitative RT-PCR for HGF and c-Met mRNAs were performed from total liver or purified cell fractions and HGF mRNA was also analyzed by in situ RT-PCR on liver sections. A LPS challenge alone induced a marked up-regulation of HGF mRNA level in whole liver and isolated hepatocytes. Furthermore, when partial hepatectomy (PH) was preceded by a LPS challenge, an increase of HGF mRNA level was seen in whole liver and contrasted with a decreased level in non parenchyma! cells. These results were confirmed by in situ RT-PCR. In isolated hepatocytes from endotoxemic rats, the mRNA level for the LPS-specific membranous receptor mCD14 was markedly up-regulated and even more so when LPS was followed by PH. Moreover, a TNFα challenge alone induced an up-regulation of HGF mRNA in hepatocytes and a down-regulation in non parenchymal cells (NPCs). Overall, when a LPS challenge is given prior to PH the major source of hepatic HGF appears to be the hepatocyte itself rather than NPCs. An autocrine HGF/c-Met loop which promotes the proliferative potential of the hepatic parenchymal cell and participates in liver regeneration is postulated.

Background: glutamine modulates cytokine production by immune cells in vitro and protects the gut from experimental enterocolitis, but data on the effect of glutamine on cytokine production in human gut are lacking. Aim: to assess the effect of glutamine pre-treatment in vivo and in vitro on cytokine production by intestinal mucosa. Methods: nine fasted volunteers received either enteral glutamine or saline over 6h in a cross-over design. Duodenal biopsies were cultured for 24h with or without glutamine. Cytokine content of culture media was analysed by ELISA, and the expression of cytokine mRNA in biopsies was assessed by semi-quantitative RT-PCR. Results: glutamine given in vivo and in vitro significantly decreased IL-6 [1.4 (0.8–8.5) vs 8.9 (1.0–43.9)] and IL-8 production [5.8 (0–51.4) vs 53.0 (2.5–114.6), pg/mg wet tissue], median (range), both P≤0.01, in comparison to no glutamine experiments. Glutamine did not influence IL-4 production. IL-1β, IL-10 and TNF-α were not detectable in culture media. The expression of any cytokine mRNA was not influenced by glutamine. Conclusions: glutamine reduces pro-inflammatory cytokine production by human intestinal mucosa, probably by a post-transcriptional pathway. Glutamine could be useful to modulate inflammatory conditions with imbalanced cytokine production.

A set of orthologous plasma proteins found in human, sheep, pig, cow and rodents, now collectively designated fetuin-A, constitutes the fetuin family. Fetuin-A has been identified as a major protein during fetal life and is also involved in important functions such as inhibition of the insulin receptor tyrosine kinase activity, protease inhibitory activities and development-associated regulation of calcium metabolism and osteogenesis. Furthermore, fetuin-A is a key partner in the recovery phase of an acute inflammatory response. We now describe a second protein of the fetuin family, called fetuin-B, which is found at least in human and rodents. On grounds of domain homology, overall conservation of cysteine residues and chromosomal assignments of the corresponding genes in these species, fetuin-B is unambiguously a paralogue of fetuin-A. Yet, fetuin-A and fetuin-B exhibit significant differences at the amino acid sequence level, notably including variations with respect to the archetypal fetuin-specific signature. Differences and similarities in terms of gene regulation were also observed. Indeed, studies performed during development in rat and mouse showed for the first time high expression of a member of the fetuin family in adulthood, as shown with the fetuin-B mRNA in rat. However, like its fetuin-A counterpart, the fetuin-B mRNA level is down-regulated during the acute phase of experimentally induced inflammation in rat.

Nowadays the design of complex systems requires the cooperation of several teams belonging to different cultures and using different languages. It is necessary to dispose of new design and verification methods to handle multilanguage approaches. This paper presents an approach for the interlanguage communication synthesis of heterogeneous specifications. The system is represented by a set of interconnected subsystems specified in different languages with different concepts, different interface types, different communication schemes and some of them can even be IP modules. The subsystems exchange data through abstract communication channels. The objective is to refine the abstract communication channels into an implementation. The result is a set of interconnected processors communicating through signals, buses and dedicated components. An example illustrates the usefulness of this approach for the design of an adaptive speed control system that was described in SDL and Matlab.

Association of kappa light chain immunoglobulin allotypes with bullous pemphigoid was examined in 101 Caucasian patients. Km alleles were determined by polymerase chain reaction amplification followed by restriction enzyme digestion. The frequency of Km3/Km1,2kappa light-chain genotype was found to be significantly associated with the disease, while that of the Km3homozygous genotype was significantly higher in patients with both anti-BPAG1 and anti-BPAG2 autoantibodies.

ABSTRACTIn this paper, we study magnetostatic problem. We establish a mixed formulation in magnetostatics and we show that the problem is well-posed with Brezzi-Babuska theory, an inf-sup condition is proved. A mixed Raviart-Thomas finite element method of minimal order is used to approximate the continuous problem in H(div) x L2. Numerical tests are implemented to validate the method. First, we considera sphere in a homogeneous source field then a domain in which the permeability is not constant.

Macrophage activation and the resulting inflammatory response may be a major component of tissue injury upon hypoxia and re-oxygenation. Activation of the haem oxygenase (HO)/carbon monoxide (CO) pathway may be an important regulator of the inflammatory response, through production of cyclic 3′, 5′-monophosphate (cGMP). We have assessed whether HO contributes to the increased production of the pro-inflammatory cytokines TNF-α and IL-6 in re-oxygenated rat peritoneal macrophages.

The paper is devoted to an approximation of the solution of Maxwell's equations in three-dimensional space. We present two methods which couple a finite element method inside the magnetic materials with a boundary integral method which uses Poincaré–Steklov's operator to describe the exterior domain. A computer code has been implemented for each method and a number of numerical experiments have been performed to validate each proposed methodology. Namely, we present numerical results concerning a non-linear magnetostatic problem in ℝ³. Copyright © 1999 John Wiley & Sons, Ltd.

Phorbol myristate acetate (PMA)-stimulated human monocyte-like cells (U-937) were found to synthesize the third component of complement (C3), as shown by enzyme-linked immunosorbent assay and immunoprecipitation from [35S]methionine-labelled culture supernatants. C3 synthesis occurred at a rate of about 160 ng of C3/24 h per 10(6) cells on day 7 after addition of PMA; it was blocked by cycloheximide treatment and was restored after removal of the inhibitor. SDS/polyacrylamide-gel-electrophoretic analysis of the immunoprecipitated protein showed that the size and subunit structure of the newly synthesized C3 were identical with those of plasma C3, and that a single-chain intracellular precursor was present in the cell lysates. Haemolytic assays showed that the synthesized C3 fully expressed functional activity in early culture within 4 h. After longer culture, a loss of haemolytic activity was observed. The possibility that newly secreted C3 is cleaved by U-937 cells themselves was suggested.

Experiments were designed to localize the central sites of action of melatonin involved in the control of seasonal reproduction. Ewes were exposed to long days and received microimplants of melatonin in the preoptic area (n = 9), anterior hypothalamus (n = 4), dorsolateral hypothalamus (n = 4), or mediobasal hypothalamus (n = 12). The effects of implants were determined by comparison with control ewes (untreated or sham-operated, n = 10) and with ewes treated with an s.c. implant of melatonin (n = 8) or ewes subjected to short days (n = 8). All ewes were ovariectomized and treated s.c. with a silastic capsule of estradiol. Melatonin released in the preoptic area as well as in the anterior and lateral hypothalamus did not cause any difference as compared with the controls (no stimulation of LH secretion and no inhibition of prolactin secretion). In contrast, melatonin implanted in the mediobasal hypothalamus caused an increase in LH secretion in 7 of the 12 ewes on Day 53.0 ± 4.2 after implantation (mean ± SEM). Their response was not different compared with that of ewes treated s.c. with melatonin or exposed to short days either in terms of timing (Day 56.3 ± 6.2 and 59.5 ± 3.1, respectively, for controls) or of amplitude of the LH response. Similarly, melatonin caused only a reduction of prolactin secretion in the mediobasal, s.c., and short-day groups. It is concluded that the mediobasal hypothalamus or the surrounding tissues could be the sites of action of melatonin involved in the control of seasonal reproduction.

This paper presents the main co-design concepts of distributed embedded hardware/software systems through a detailed presentation of a methodology and an environment for codesign. This presentation gives a comprehensive description of different codesign steps using a codesign of a realistic example: Real-Time Acquisition and Storage Controller acting between analog signals and storage disk. Design starts from a system-level specification and performs hardware/software partitioning, communication synthesis and architecture generation. To produce an efficient solution, the codesign process is guided by performance and constraints that depend on the application context.

The family of plasma proteins collectively referred to as Inter-α-Inhibitor (IαI) family is comprised of a set of multi-polypeptide molecules and a single-chain molecule designated IαIH4P. Although the 4 heavy chain precursors H1P to H4P that lead to these molecules are evolutionarily related, only H4P harbours a Pro-rich region (PRR) in its C-terminal third. A comparison of hepatic H4P cDNAs in human and rat has now unraveled an extensive variability of this PRR. Within the rat PRR, 6 repeats of a Gly-X-Pro motif participate in a collagen-like pattern that is absent in human. Within the human PRR, a domain that is absent in rat can be transcribed or deleted by alternative splicing which results in two variant forms of human H4P. In rat liver, the single mRNA is up-regulated by an acute, systemic inflammation whereas neither mRNA is up-regulated in human liver. Finally the shortest human mRNA is also transcribed in peripheral blood mononuclear cells where it is down-regulated by bacterial lipopolysaccharides. Therefore, in contrast to what is seen for theITIH1to-3genes, the rat and humanITIH4gene transcriptions and products thereof present marked differences, which suggests species-specific functions for IαIH4P.

Serotonin and serotonin receptors of class II (5HT2-R) are thought to be involved in the neural mechanisms which regulate the LH release associated with photoperiodic changes in sheep. A specific premammillary hypothalamic area displaying a significant binding of ³H-ketanserin, a potent 5HT2-R antagonist, was previously identified. The aim of the present study was to ascertain by in situ hybridization (ISH) that 5HT2-R mRNA-containing cells were also present in this specific hypothalamic area. Total RNA was prepared from sheep pars tuberalis/median eminence, and a cDNA fragment of 546 bp was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerated primers deduced from the human and rat 5HT2A-R sequences. After cloning and sequencing, the sheep nucleotide sequence had the highest homology (85.1-92.3%) with the other known mammalian 5HT2-R or 5HT2A-R sequences. Homology with other 5HT-R subtypes or other monoamine receptors was much lower, 60% at maximum. After ISH using sense and antisense ³⁵S-riboprobes, specific labelling was found in different parts of the hypothalamus, especially in the mammillary bodies where the binding was higher. Within the hypothalamus, the density of labelled cells, mainly neurons, varied considerably. It was maximal in the mammillary bodies and also in a restricted ventral region of the premammillary hypothalamus located from about 500/700 m to 1200/1400 m in front of the mammillary recess, where ³H-ketanserin binding was previously reported. In conclusion: (1) the structural study of the sequence indicated that the new cloned cDNA corresponds to the sheep 5HT2-R class and, probably, to the 5HT2A-R subtype and (2) the ISH studies revealed that a restricted area of the premammillary hypothalamus shows a large number of 5HT2-R mRNA-containing neurons.

The inter-alpha-inhibitor (I alpha I) family encompasses four plasma proteins, namely free bikunin as well as I alpha I, pre-alpha-inhibitor (P alpha I) and inter-alpha-like inhibitor (I alpha LI). Each of the last three proteins is a distinct assembly of one bikunin chain with one or more unique heavy (H) chains designated H1, H2 and H3. The three H chains and the bikunin chain are encoded by four distinct mRNAs. These molecules and chains, as well as the corresponding mRNAs, were quantified in sera and liver biopsies from a series of patients with or without mild or severe acute infection. The decrease or increase observed for a given molecule or chain in the serum was in agreement with a similar change in the corresponding liver mRNA. In acute inflammation the H2 and bikunin chains are down-regulated and the relevant molecules (I alpha I, I alpha LI) behave as negative acute-phase proteins, whereas the H3 chain is up-regulated and the corresponding P alpha I molecule is a positive acute-phase protein. Also, P alpha I displays a higher-than-usual M(r); this is probably due to ligand binding. The H1 gene does not seem to be affected by the inflammatory condition. The quantitative changes in RNA levels seen in vivo were confirmed in vitro in the human hepatoma Hep3B cell line prior to or after induction with the acute-phase mediators interleukin-1 and/or -6. These results provide the first example in humans of positive and negative acute-phase proteins that are encoded by evolutionary related genes.

The family study of some IgG subclasses and Gm quantitative expression was carried out on sera from members of 61 families where at least one subject was affected by a chromosomal abnormality.

Screening of a human liver cDNA expression library with specific anticeruloplasmin antibodies allowed the identification of a 3' Eco RI cDNA fragment larger (+ 0.2 Kb) than the previously described cDNAs. Sequencing of this fragment revealed that it possessed, respectively, 128 and 198 additional bp when compared to published data. PCR and hybridization experiments indicated that the additional 0.2 Kb piece belonged to the Cp gene. In addition, results from Southern hybridization of DNA digests and Northern hybridization with mRNA from normal individuals, using this 0,2 Kb additional piece, suggest that the Cp gene may be duplicated on chromosome 3. J. Trace Elem. Exp. Med. 12:287–295, 1999. © 1999 Wiley-Liss, Inc.

The regulation of the synthesis of α-2-HS glycoprotein (AHSG) by inflammatory mediators from activated monocytes was studied on the human hepatoma cell line HepG2 and compared to that of albumin. Monocyte-conditioned medium, recombinant human interleukin-6 (rhIL6) and interleukin-1β(rhIL1β) all down-regulated the synthesis of AHSG. This decrease was found both at the protein and the mRNA level. The most efficient mediator was the monocyte-conditioned medium, when rhIL1β was found to be less efficient than rhIL6. The combination of rhIL6 and rhIL1β resulted in an additive down-regulation of the AHSG mRNA levels. Similar results were obtained with albumin. These data indicate that AHSG is a negative acute-phase protein whose synthesis is regulated by cytokines in a manner similar to that of albumin.

The serum level of IL-6 and expression of IL-6 mRNA in hepatocytes from regenerating liver were investigated in the rat. The IL-6 level in the serum was not significantly different from that of a control group of rats submitted to an acute experimental inflammation. IL-6 mRNA expression did not occur in the liver of hepatectomized rats as judged from Northern blotting experiments using an IL-6 riboprobe. These results suggest that if IL-6 is implicated in hepatic regeneration, this cytokine is not produced by the regenerating liver and must be delivered exogenously to the liver to modulate hepatic regeneration.

The effect of glutamine on the production of interleukin‐6 (IL‐6) was studied in rat peritoneal macrophages in culture. A maximal production of IL‐6 was measured at 4 h in lipopolysaccharide (LPS)‐stimulated macrophages, and addition of glutamine (5 mM) anticipated this increase by 1 h without any increase in the IL‐6 mRNA level. The effect of glutamine required the presence of LPS. Thus, glutamine accelerates IL‐6 production from the pre‐existing mRNA. The effect of glutamine was not mediated by cell swelling since culture of macrophages in hypoosmotic condition decreased the production of IL‐6 in the culture medium with a corresponding decrease in the IL‐6 mRNA level.

Pro-inflammatory cytokines are produced after systemic or local inflammation by a wide variety of cell types including monocytes, macrophages, Kupffer and endothelial cells. Previous studies have shown that IL-6 gene expression does not occur in liver from rats undergoing an acute phase response after turpentine injection or controls. These data do not rule out the possibility that delivery of a pathogen to the liver via the portal circulation could directly activate the Kupffer cells. Rats were injected either intravenously or intraperitoneally with LPS, or subcutaneously with turpentine oil. The changes in IL-1β, IL-6, and TNF mRNA levels in monocytes (collected from portal vein or caval cein), peritoneal macrophages and liver over a 3-hour period post-treatment were examined. The kinetics of LPS- vs turpentine-induced cytokine mRNAs in these various cell types were compared by quantitative reverse transcription and polymerase chain reaction (RT-PCR). Our data demonstrate that an intrahepatic expression of cytokines in the non parenchymal cells was induced by an LPS challenge but not by a turpentine-induced inflammation. This process could act as a paracrine mechanism in the acute-phase response and play a role in the modulation of hepatic regeneration.

Inter-α-inhibitor (IαI) and related molecules, collectively referred to as the IαI family, are a group of plasma protease inhibitors. They display attractive features such as precursor polypeptides that give rise to mature chains with quite distinct fates and functions, and inter-chain glycosaminoglycan bonds within the various molecules. The discovery of an ever growing number of such molecules has raised pertinent questions about their pathophysiological functions. The knowledge of this family has long been structure-oriented, whereas the structure/function and structure/regulation relationships of the family members and their genes have been largely ignored. These relationships are now being elucidated in events such as gene transcription, precursor processing, changes in plasma protein levels in health and disease and binding capacities that involve hyaluronan as well as other plasma proteins as ligands. This review presents some recent progress made in these fields that paves the way for an understanding of the functions of IαI family members in vivo. Finally, given the wealth of heterogeneous, complicated and sometimes contradictory nomenclatures and acronyms currently in use for this family, a new, uniform, nomenclature is proposed for IαI family genes, precursor polypeptides and assembled proteins.

The regulation of the synthesis by the cytokines interleukin-1 (IL-1) and IL-6 of the positive acute-phase protein alpha 1-acid glycoprotein (AGP) and of the negative acute-phase protein alpha 2-HS glycoprotein (AHSG) has been studied in a long-term culture system of the human hepatoma cell line Hep3B. The culture system contained 30 nM-sodium selenite as the only supplement. This allowed maintenance of the synthesis of the proteins under study at a near steady state for over 3 months. An increase in AGP mRNA and a decrease in AHSG mRNA were observed when cells were treated for two successive 48 h-periods with monocyte-conditioned medium. A return to basal levels was obtained after cessation of the cytokine addition. Two further additions of cytokines led to alterations in mRNA levels similar to those observed following the first cytokine treatment. The amounts of AGP and AHSG secreted were altered in accordance with the mRNA modifications. These results suggest that new cytokine receptors were being constantly synthesized during cell culture. When cytokines were present in the culture medium for 10 days, maximum alterations in AGP and AHSG synthesis were obtained following 2 and 4 days of treatment respectively, but further alterations in protein levels could not be observed afterwards. Expression of IL-6 receptor mRNA was not up-regulated by cytokines, but only by 1 microM-dexamethasone. Our results show that, in this long-term culture system, cytokines induce a response in hepatoma cells similar to that observed in vivo during human inflammatory states. This model could be used to evaluate the effects of agonists or antagonists of cytokines responsible for the hepatic acute-phase protein response.

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.

We used a system of co-culture of adult rat hepatocytes with another epithelial cell type from rat liver to study the synthesis of two acute-phase reactants, alpha-1 acid glycoprotein (alpha 1AGP) and the third component of complement (C3), and we have obtained long-term secretion of these two proteins. After a period of adaptation corresponding to the first 2-4 days of the co-culture, hepatocytes secreted C3 and alpha 1AGP for at least 2 weeks at a mean level higher than that observed in the first days of a pure culture of hepatocytes. When pulse-chase analysis was performed on day 6 of co-culture, kinetics of synthesis of alpha 1AGP and C3 were the same as those observed on day 1 of a conventional culture of pure hepatocytes. Furthermore, intracellular and extracellular alpha 1AGP had Mr values respectively of 39,000 and of 42,000-52,000, identical with those observed in pure cultures of hepatocytes. Similarly, the molecular size and subunit structures of C3 were the same in co-culture and in cultures, indicating an identical processing of this protein. C3 produced in co-culture was also haemolytically active. Therefore, the system of adult hepatocytes co-cultured with this liver epithelial cell provides a physiological system in vitro which permits long-term synthesis of the two acute-phase reactants C3 and alpha 1AGP. This model opens the possibility to study the modulation of the synthesis of these two proteins during a long period by inflammatory agents or by hormones.

The expression and level of the mRNAs for the five genes that code for a set of plasma proteins collectively referred to as the inter-α-inhibitor family have been studied in rat under a normal condition or in the course of a turpentine-induced, systemic inflammation. In healthy rats, all five mRNAs [H1, H2, H3, H4, and α1-microglobulin/bikunin precursor (AMBP)] are expressed primarily in liver and two of them (H2 and H3) are found to a lower extent in brain. Byin situhybridization onto sections of a normal brain, the H3 mRNA has been precisely localized to the hypothalamus, amygdala, pontine area, optic tectum, and cerebellum. By reverse transcriptase-polymerase chain reaction of total RNAs obtained from a panel of organs, low amounts of one or more mRNA(s) could be detected in other locations (e.g., intestine and stomach). Furthermore, the extrahepatic expressions of several of these genes are up- or downregulated at 20 h after the start of a turpentine-induced inflammation. In liver, the contents of H3 and H4 mRNA are upregulated, whereas those of AMBP and H2 are downregulated during the acute phase. This is accounted for by changes in gene transcription, the kinetics of which is gene-specific. This behavior of H1, H2, H3, H4, and AMBP mRNAs in rat liver is in keeping with more limited analyses made at mRNA and/or protein levels in other species (human, pig) suffering from an acute inflammation. Therefore, the inflammation-associated regulation of these five genes that is conserved between species indicates that the inter-α-inhibitor family members are likely to be important partners of the acute phase response.

Summary.The time of appearance of plasma LH and testosterone peaks through the day was determined in 75 Préalpes du Sud and 41 Ile-de-France rams in December and in 44 Préalpes du Sud and 11 Ile-de-France rams in June. The distribution of peaks throughout the day was non-random for the two hormones in the two breeds and for both times of the year (P< 0·01 at least on each occasion; P< 0·001 on pooled data from the two breeds). The most striking features were the occurrence of (1) a minimum of LH and testosterone peaks immediately after 'dawn' (lights on) in both months; (2) a maximum of peaks 3 h after 'dawn' in June and 4 h after 'dawn' in December.For several hours after the increase in frequency of peaks the probability of measuring peaks of LH and testosterone remains high. This and the correlation between the LH values in December and June when adjusted for the time of 'dawn' suggest that dawn could act as a synchronizer of gonadotroph activity.