Biochemical actions of chronic ethanol exposure in the mesolimbic dopamine system

In previous studies, we have demonstrated that chronic administration of morphine or cocaine produces some common biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc), components of the mesolimbic dopamine system implicated in the reinforcing actions of these and other drugs of abuse. Since this neural pathway is also implicated in the reinforcing actions of ethanol, it was of interest to determine whether chronic ethanol exposure results in similar biochemical adaptations. Indeed, as seen for chronic morphine and cocaine treatments, we show here that chronic ethanol treatment increased levels of tyrosine hydroxylase and glial fibrillary acidic protein immunoreactivity, and decreases levels of neurofilament protein immunoreactivity, in the VTA. Also like morphine and cocaine, ethanol increases levels of cyclic AMP-dependent protein kinase activity in the NAc. These actions of ethanol required long-term exposure to the drug, and were in most cases not seen in the substantia nigra or caudate-putamen, components of the nigrostriatal dopamine system studied for comparison. Altered levels of tyrosine hydroxylase in catecholaminergic cells frequently reflect altered states of activation of the cells. Moreover, increasing evidence indicates that ethanol produces many of its acute effects on the brain by regulating NMDA glutamate and GAB_A receptors. We therefore examined the influence of chronic ethanol treatment on levels of expression of specific glutamate and GABA receptor subunits in the VTA. It was found that long-term, but not short-term, ethanol exposure increased levels of immunoreactivity of the NMDARl subunit, an obligatory component of NMDA glutamate receptors, and of the Glu Rl subunit, a component of many AMPA glutamate receptors; but at the same time, long-term ethanol exposure decreased immunoreactivity levels of the α1 subunit of the GABA_A receptor complex. These changes are consistent with an increased state of activation of VTA neurons inferred from the observed increase intyrosine hydroxylase (TH) expression. These results demonstrate that chronic ethanol exposure results in several biochemical adaptations in the mesolimbic dopamine system, which may underlie prominent changes in the structural and functional properties of this neural pathway related to alcohol abuse and alcoholism. © 1995 Wiley-Liss, Inc.     

Définition et évolution du statut nutritionnel au cours d'une greffe osseuse allogénique (15 malades): Rôle de la nutrition parentérale et de l'apport azoté

Our work concerned 15 patients (9 males, 6 females) with a mean age of 29.5 years, having a hematologic malignant disease and undergoing allogenic bone marrow transplantation.We studied :

1. The metabolic disorders induced by the conditioning regimen (chemotherapy and total body irradiation) pregraft accompanying cytolysis (day −7, −5, −2).

2. The corrective effect of a total parenteral nutrition introduced 2 days before the transplantation and pursued during 30 days post-graft (day −2 to day 30).

3. The interest of a high calorie intake (BEE × 2) and, after randomisation, of a variable nitrogen intake (24% of the total calorie intake for group A [8 patients] and 14% for group B [7 patients]). The patient characteristics of these two groups were closely comparable. Urinary parameters were studied daily (3-methylhistidine, cratinine, nitrogen) and blood parameters weekly (transferrin, pre-albumin, albumin, retinol binding protein).

We observed globally :

-- An excellent result of the nutritional support without significant weight loss;

-- protein catabolism stopped with a recovery of synthesis of RBP after day 7 and pre-albumin from day 7;

-- a decrease in muscle catabolism.

The randomized study showed :

-- a significant difference in nitrogen excretion between group A and group B;

-- earlier and better protein synthesis recovery in group A, particularly with regard to RBP and pre-albumin.

In conclusion, we recommend for the patients undergoing bone marrow transplantation :

-- nutritional support should be introduced before the conditioning regimen;

-- a high calorie intake (BEE × 2) with a nitrogen intake between 14% and 24% of the total calorie intake;

-- cyclic parenteral nutrition should be pursued during the second and third month post-graft.

Inflammatory responses in blood samples of human immunodeficiency virus-infected patients with pulmonary infections

We analyzed the characteristics of the inflammatory response occurring in blood during pulmonary infections in human immunodeficiency virus (HIV)-infected patients. A prospective study of consecutive hospital admissions of HIV-infected patients with new-onset radiologic pulmonary infiltrates was carried out in a tertiary university hospital from April 1998 to May 2001. Plasma cyclic AMP receptor protein (CRP), interleukin 1beta (IL-1beta), IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNF-alpha) levels were determined at the time of admission and 4, 5, and 6 days later. Patients were included in a protocol addressed to study etiology and outcome of disease. A total of 249 episodes of infection were included, with the main diagnoses being bacterial pneumonia (BP) (118 episodes), Pneumocystis carinii pneumonia (PCP) (41 episodes), and mycobacteriosis (36 episodes). For these three patient groups, at the time of admission the median CRP and cytokine levels were as follows: CRP, 10.2, 3.8 and 5 mg/dl, respectively (P = 0.0001); IL-8, 19, 3, and 2.9 pg/ml (P = 0.045); and TNF-alpha, 46.4, 44, and 75 pg/ml, respectively (P = 0.029). There were no significant differences in levels of IL-1beta, IL-6, or IL-10 among the patient groups. A total of 23 patients died. At the time of admission, HIV-infected patients with BP had higher plasma CRP and IL-8 levels than did PCP and mycobacteriosis patients. TNF-alpha levels were higher in patients with mycobacteriosis. An elevated IL-8 level (>61 pg/ml) at the time of admission was an independent factor associated with higher mortality (odds ratio, 12; 95% confidence interval, 1.2 to 235.5).     

Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation

Cytokine-producing cells in gut-associated lymphoid tissues of rhesus macaques with chronic enterocolitis were studied. The confocal microscopy technique that we developed enables simultaneous in situ visualization of multiple extra- and/or intracellular antigens at a resolution higher than that allowed by light or epifluorescence microscopy. The presence of interleukin-6 (IL-6)-, tumor necrosis factor alpha (TNF-alpha)-, and IL-1-alpha-producing cells was focally intense in the colon lamina propria of the affected animals. The IL-1-alpha-producing cells were T lymphocytes (CD3+), while the TNF-alpha-producing cells were both macrophages (CD68+/HAM56+/LN5+) and T lymphocytes (CD3+). The IL-6-producing cells within the colon consisted of T lymphocytes and macrophages. The amount of IL-6-producing cells seen in macaques with enterocolitis was significantly higher (P<0.001) than that seen in the healthy control animal, while TNF-alpha- and IL-1-alpha-producing cells were seen only in macaques with enterocolitis. Most of the T lymphocytes that produced cytokines were detected in the lamina propria, while the macrophages were most prominent in highly inflamed regions of the lamina propria. Taken together, our findings indicate that there might be immunological similarity between chronic enterocolitis of rhesus macaques and humans, suggesting the potential use of the nonhuman primate model for the validation of novel therapies.     

Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.     

Mutation analysis of the SDHD gene in four kindreds with familial paraganglioma: description of one novel germline mutation.

The familial paraganglioma syndrome is an autosomal dominant disorder characterized by the presence of carotid body paragangliomas and, less frequently, paragangliomas of the glomus jugulare, glomus vagale, and adrenal pheochromocytomas. Germline mutations of the genes for succinate dehydrogenase subunits D, B, or C (SDHD, SDHB, SDHC) have been identified in some kindreds with familial paraganglioma. In this study, we report the clinicopathologic features of four different kindreds with familial paraganglioma, which were screened for germline mutations in the SDHD gene. DNA was obtained from tumor and normal tissue, as well as from peripheral blood. Mutation analysis was performed by single-strand conformation polymorphism analysis and DNA sequencing. SDHD germline mutations were detected in the affected family members of the four families, as well as in several asymptomatic carriers. An identical mutation in exon 4 of SDHD (334-337delACTG) was identified in two apparently unrelated kindreds. The third family showed a germline mutation in exon 2 (W43X). The mutations present in these three families had been previously described in Spanish families, suggesting a founder effect. The fourth family exhibited a mutation in exon 2 of SDHD (170-171delTT), which had not been previously identified. The affected family members of the four kindreds showed paragangliomas, located in the head and neck region, and all of them were benign. These results confirm that genetic testing of SDHD may be a powerful tool for the identification of the syndrome in patients with multiple or bilateral paragangliomas.     

Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.     

Autoantibodies directed against the amino-terminal domain I of human calpastatin (ACAST-DI Ab) in connective tissue diseases. High levels of ACAST-DI Ab are associated with vasculitis in lupus

To identify new autoantibody populations in patients with rheumatic diseases, a cDNA expression library was immunoscreened with a rheumatoid arthritis (RA) patient's serum which contains autoantibodies binding to uncharacterized polypeptides by Western-blotting. One clone encoding the amino-terminal region (Nt) [domain L and half of domain I] of human calpastatin was selected. Different fragments of the selected cDNA were prepared and the corresponding recombinant polypeptides were produced by in vitro translation and analysed by Western blotting. Most RA sera bound to recombinant amino-terminal region and domain I but not to domain L. This prompted us to use a recombinant polypeptide corresponding to the domain I of calpastatin as the antigen in a solid-phase ELISA to test sera from patients with various systemic rheumatic diseases and healthy controls.Anti-calpastatin domain I antibodies (ACAST-DI Ab), were detected by ELISA in RA, systemic lupus erythematosus (SLE), Sj?gren's syndrome and control sera at respective frequencies of 10, 9, 0 and 1%. These Ab did not have prognostic value in early RA; high levels were significantly associated with vasculitis in SLE. Antibodies reacting with the calpastatin amino-terminal region are produced during systemic rheumatic diseases and are predominantly directed against domain I. High levels of these Ab may constitute a marker of vasculitis in SLE.     

Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.