HLA-G*01:04∼UTR3 Recipient Correlates With Lower Survival and Higher Frequency of Chronic Rejection After Lung Transplantation.

Lung transplantation (LTx) is a valid therapeutic option for selected patients with end-stage lung disease. Soluble HLA-G (sHLA-G) has been associated with increased graft survival and decreased rejection episodes in solid organ transplantation. HLA-G haplotypes named UTRs, defined by SNPs from both the 5'URR and 3'UTR, have been reported to reliably predict sHLA-G level. The aim of this retrospective study was to determine the impact of HLA-G alleles and UTR polymorphism from LTx recipients on anti-HLA allo-immunization risk, overall survival and chronic rejection (CLAD). HLA-G SNPs were genotyped in 124 recipients who underwent LTx from 1996 to 2010 in Marseille, 123 healthy individuals and 26 cystic fibrosis patients not requiring LTx. sHLA-G levels were measured for 38 LTx patients at D0, M3 and M12 and for 123 healthy donors. HLA-G*01:06∼UTR2 was associated with a worse evolution of cystic fibrosis (p = 0.005) but not of long-term survival post-LTx. HLA-G*01:04∼UTR3 haplotype was associated with lower levels of sHLA-G at D0 and M3 (p = 0.03), impaired long-term survival (p = 0.001), increased CLAD occurrence (p = 0.03) and the production of de novo donor-specific antibodies (DSA) at M3 (p = 0.01). This study is the first to show the deleterious association of different HLA-G alleles and UTRs in LTx.

Alexithymia and eating behaviour in severely obese patients.

BACKGROUND: Alexithymia is the inability to express feelings with words and comprises a psychological construct frequently found in obese individuals. In eating disordered patients who show a tendency to lose control over food intake, personality traits with alexithymic characteristics have been demonstrated. The present cross-sectional study investigated the relationships between alexithymia and eating behaviour in severely obese patients. METHODS: This study analysed 150 obese patients undergoing bariatric surgery and 132 subjects at more than 1 year after biliopancreatic diversion (BPD), when body weight has steadily normalised and any preoccupation with weight, food and diet has been completely abandoned. Obese and operated subjects completed the Toronto Alexithymia Scale (TAS), and eating behaviour was assessed via a semi-structured interview exploring binge eating disorder (BED), night eating and emotional eating, as well as by utilisation of the Three Factor Eating Questionnaire (TFEQ). RESULTS: Although alexithymic patients showed deranged eating behaviour, as evaluated by the TFEQ scores, the frequency of BED, night eating and emotional eating was similar in alexithymic (TAS > 60) and non-alexithymic patients. However, the prevalence of alexithymia was similar in obese and BPD subjects, whereas, in the operated subjects, TFEQ scores were lower (P < 0.005) than those in obese patients. CONCLUSIONS: These data suggest that, in severely obese patients, alexithymia does not influence eating behaviour; in severely obese patients, the tendency to lose control over food intake apparently represents a psychological construct that is substantially independent from alexithymia.

Pouch diverticula after vertical-banded gastroplasty.

PURPOSE: Diverticula of the proximal gastric pouch are rare after vertical-banded gastroplasty (VBG) for morbid obesity. We report the radiographic findings observed in a series of 12 patients with pouch diverticula. MATERIALS AND METHODS: Lesions were found along the posteromedial wall of the proximal gastric pouch and ranged in size from 10 to 25 mm. Only two patients were symptomatic at the time of diagnosis; in most cases, diverticula were discovered during studies performed as part of the standard follow-up protocol. Diverticula were followed up in 7/12 cases, and four showed slight enlargement over a period ranging from 14 to 53 months. RESULTS: The presence of diverticula was not correlated with symptoms, postoperative weight loss, or clinical history, and no differences in long-term complications were demonstrated between VBG patients with diverticula and those without them. CONCLUSIONS: We do not believe these lesions to be clinically important; at present, our patients are no longer followed up for this problem and undergo diagnostic examinations only if and when they develop symptoms.

[Laparoscopic liver surgery for metastases of colorectal cancer: analysis of a monocentric experience]. / Laparoscopic liver surgery for metastases of colorectal cancer: analysis of a monocentric experience.

BACKGROUND: Advances in laparoscopic techniques, refinements of instruments and growth of practical experience in liver surgery during the last decade have prompted some surgeons to develop the laparoscopic approach for hepatic metastases of colorectal cancer (MCRC). AIMS: Primary end points of this clinical study were safety and effectiveness of laparoscopic hepatectomy for MCRC, including early postoperative results and long-term outcomes (overall survival and disease-free survival). DESIGN: Retrospective analysis of data (clinicopathologic, operative, perioperative ad late results) collected in a prospective database. PATIENTS: Between January 1997 and December 2004, 37 non-consecutive (selected) patients underwent curative laparoscopic hepatic resection (n = 42) for MCRC at Montsouris Institut of Paris. Resection was considered when all liver metastases can be totally removed with clear margins, and in absence of nonresectable extrahepatic diseases. Among them were 24 males and 13 females with average ages of 63.4 years (range, 42-78). RESULTS: Metastases were metachronous in 18, multiple in 21, bilateral in 12, and <5 cm in diameter in 30. There were 21 major hepatectomies (n = 3 Couinaud's segments or more), 4 anatomical minor resections, and 12 wedge resections. Mean operative time was 324 +/- 105 mins. Conversion to laparotomy was necessary in 6 patients (16%), due to massive intractable bleeding in 3 patients, multiples adhesions in 1 patient, technical reasons (location of the lesion) in 1 patient, and for presence of localized carcinosis in 1 patient. Portal triad clamping was performed in 6 patients. Mean operative blood loss was 797 +/- 645 ml, and transfusions were required in 4 patients (11%). Clear resection margins (> 5 mm) were observed in 94%. Postoperative mortality was nil. The overall morbidity rate was 35%, with 2 early reoperations due to hemorrhage and postoperative ileus. Overall and disease free survival at 36 months were 87% and 55%, respectively. Five patients who had a recurrence of metastatic liver disease were referred to a second laparoscopic resection. CONCLUSION: This clinical study suggests that laparoscopic liver surgery for metastatic colorectal cancer can be accomplished safely, in selected patients and by experienced surgeons, with good early results and without detrimental consequences on survival.

Reactivation and persistence of human herpesvirus-8 infection in B cells and monocytes by Th-1 cytokines increased in Kaposi's sarcoma.

Patients with Kaposi's sarcoma (KS) have a human herpesvirus-8 (HHV-8) load higher than patients without KS and present a CD8(+) T-cell activation with production of Th1-type cytokines both in tissues and peripheral blood mononuclear cells (PBMC). Because in tissues of KS patients detection of inflammatory cytokines (IC) can precede detection of HHV-8 DNA and because signs of immunoactivation and/or dysregulation can precede KS development, we investigated the effect of IC on HHV-8 infection. To achieve this goal, PBMC and purified cell populations from 45 patients with KS and 45 patients at risk of KS were analyzed for HHV-8 DNA and/or gene expression and for cell survival, growth, and phenotype before or after culture with or without the IC increased in KS. The results indicate that PBMC that are polymerase chain reaction (PCR)-positive at day 0 generally loose the virus upon culture. However, the presence of IC maintains HHV-8 DNA load in cultured cells. In addition, IC increase viral load to detectable levels in PBMC from serologically positive patients that were PCR-negative before culture. gamma Interferon is sufficient for these effects, whereas tumor necrosis factor and interleukin-6 have little or no activity. The increase of HHV-8 DNA by IC is observed after short-term (7 days) or long-term (28 days) culture of the cells and occurs in one or both of the two circulating cell types that are infected in vivo: B cells and monocytes. In both cases it is associated with lytic gene expression, suggesting that virus reactivation is one of the most likely mechanisms for the effect of IC on virus load. However, IC have also effects on the cells target of HHV-8 infection, because they increase B-cell survival and induce the growth and differentiation of monocytes into KS-like spindle cells with markers of endothelial macrophages. Because cells with markers of endothelial macrophages are present in blood and lesions from KS patients and are infected by HHV-8, these data may explain the high HHV-8 load associated with KS development and suggest that infected monocytes may carry the virus to tissues, transmit the infection, or differentiate in loco in spindle cells with endothelial macrophage markers.

Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells.

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.

Reversion of a human immunodeficiency virus type 1 integrase mutant at a second site restores enzyme function and virus infectivity.

The integration of a DNA copy of the retroviral RNA genome into the host cell genome is essential for viral replication. The virion-associated integrase protein, encoded by the 3' end of the viral pol gene, is required for integration. Stable virus-producing T-cell lines were established for replication-defective human immunodeficiency virus type 1 carrying single amino acid substitutions at conserved residues in the catalytic domain of integrase. Phenotypically reverted virus was detected 12 weeks after transfection with the integrase mutant carrying the P-109-->S mutation (P109S). Unlike the defective P109S virus, the revertant virus (designated P109SR) grew in CD4+ SupT1 cells. In addition to the Ser substitution at Pro-109, P109SR had a second substitution of Ala for Thr at position 125 in integrase. Site-directed mutagenesis was used to show that the P109S T125A genotype was responsible for the P109SR replication phenotype. The T125A substitution also rescued the in vitro enzyme activities of recombinant P109S integrase protein. P109S integrase did not display detectable 3' processing or DNA strand transfer activity, although 5 to 10% of wild-type disintegration activity was detected. P109S T125A integrase displayed nearly wild-type levels of 3' processing, DNA strand transfer, and disintegration activities, confirming that T125A is a second-site intragenic suppressor of P109S. P109S integrase ran as a large aggregate on a size exclusion column, whereas wild-type integrase ran as a monomer and P109S T125A integrase ran as a mixed population. Pro-109 and Thr-125 are not immediately adjacent in the crystal structure of the integrase catalytic domain. We suggest that the T125A substitution restores integrase function by stabilizing a structural alteration(s) induced by the P109S mutation.

The non-producer phenotype of the human immunodeficiency virus type 1 provirus F12/HIV-1 is the result of multiple genetic variations.

A cell clone (Hut-78/F12) chronically infected with a non-producer human immunodeficiency virus type 1 (HIV-1) variant showed an abnormal pattern of virus structural proteins and released no detectable virus particles. Exchanges of homologous parts of the F12/HIV provirus and a replication-competent HIV (strain NL4-3) were undertaken to define the genetic determinants of the F12/HIV phenotype. The non-infectious phenotype was reproduced by replacing an NL4-3 genomic fragment encoding the C terminus of gp 120 and the N terminus of gp41 with the corresponding parts of the F12/HIV provirus. Conversely, a much more extended genomic fragment (encompassing the vif, pol and env genes) was necessary to convert the F12/HIV phenotype. These results demonstrate that the F12/HIV non-producer phenotype is the result of mutations scattered along most of the genome, rendering the conversion to an infectious phenotype a very unlikely event. The F12/HIV genome is thus a reliable model for preclinical studies of anti-HIV gene therapy.

A recombinant retrovirus carrying a non-producer human immunodeficiency virus (HIV) type 1 variant induces resistance to superinfecting HIV.

A human immunodeficiency virus (HIV) type 1-infected Hut-78 cell clone (F12) shows a peculiar phenotype: it exhibits an altered viral protein pattern, is a nonproducer and is resistant to homologous superinfection. To determine whether this phenotype is dependent upon the expression of the HIV-1 genome integrated therein, the SstI/SstI F12 provirus [deprived of HIV long terminal repeats (LTRs)] was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo) and Geneticin resistance gene. CD4+ HIV-susceptible CEMss cells (a CEM clone able to form large syncytia 2 to 3 days post-HIV infection) were infected with the recombinant retroviruses rescued from the F12/HIV-pLj-transfected (in either sense or antisense orientation) amphotropic packaging cells PA 317. Neo sense resistant gene clones showed approximately 10 copies of viral DNA/cell (without detectable major deletions) only in episomal form, low viral RNA expression and a viral protein pattern characterized by an uncleaved gp160, no gp41 and little, if any, p55 gag precursor (as in F12 cells). Superinfection of these F12/HIV DNA-engineered clones with HIV-1 resulted in a significant reduction in the yield of superinfecting HIV. This effect (more pronounced when the clones were maintained under neo selective pressure) was observed in all five retrovirus-infected clones exhibiting the presence and expression of sense episomal F12/HIV DNA but not in two clones bearing an antisense F12/HIV DNA or in one clone bearing only the pLj vector. These results indicate that bio-engineered human CD4+ cells expressing the F12/HIV genome exhibit a significant resistance to HIV superinfection.

Biologic and molecular characterization of producer and nonproducer clones from HUT-78 cells infected with a patient HIV isolate.

HUT-78 cells were infected with a reverse transcriptase (RT)-positive supernatant of a culture of peripheral blood lymphocytes (PBL) from an AIDS patient and then cloned. Of these clones, two have been isolated and characterized. Clone D10 is persistently and productively infected with an HIV variant. The clone F12, in spite of the presence of an integrated full-length HIV provirus, does not release virus particles in the medium. D10 and F12 clones substantially differ in terms of protein pattern; that is, D10 is super-imposable to infected HUT-78 cells, whereas F12 exhibits a decreased uncleaved p55 gag precursor and the presence of uncleaved gp160 and of a unique p19, although they do not show qualitative or quantitative differences in viral RNA synthesis. Restriction patterns of F12 proviral DNA do not show major genomic deletions. These results indicate that F12 clone cells carry an HIV genome with minor mutations that probably affect the correct production of viral proteins at a posttranscriptional level. In addition, the F12 clone is resistant to high-multiplicity superinfection with HIV-1 or HIV-2.

Purification and partial characterization of a novel thyroxine-binding protein (27K protein) from human plasma.

T4-binding globulin (TBG) prepared from human plasma by the standard three-step procedure (T4-agarose affinity chromatography, anion exchange chromatography, and gel filtration) often shows in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in addition to the expected 54K band, another with a mol wt of 27,000 (27K protein). The two proteins can be separated after the three-step procedure by chromatofocusing (because of different isoelectric points, 4.2-4.8 for TBG and 5.0-5.2 for 27K protein) or by T4-aragose chromatography eluting with a linear gradient of T4 (TBG is eluted between 10(-10) and 10(-9) M T4, 27K protein between 10(-8) and 10(-7) M T4). The 27K protein does not appear to be a fragment of TBG since 1) it does not displace [125I]TBG bound to anti-TBG monoclonal antibodies; and 2) absorption of polyclonal antibody reacting with both TBG and 27K protein with sera from TBG-deficient patients completely prevents [125I]27K protein binding, while only slightly affecting [125I]TBG binding. On the other hand, 27K protein is not simply a contaminant devoid of biological activity, but is a T4-binding protein, as supported by the following findings: 1) it covalently binds [125I]T4 by photoaffinity labeling, and this binding can be almost completely prevented by excess T4; 2) equilibrium dialysis shows two equivalent T4-binding sites per 66K, with an association constant of 0.85 X 10(7) M-1, intermediate between albumin and prealbumin; and 3) tryptophanyl fluorescence analysis shows quenching of 37% of the fluorescence when the protein is titrated with T4. The 27K protein appears as a single 27K band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pH 8.8, but under nondenaturing nonreducing conditions mostly remains at the origin of the gel; a fraction enters the gel and migrates slightly ahead of albumin. This electrophoretic pattern is distinct from those of albumin, prealbumin, and TBG. In immunoelectrophoresis in agar at pH 8.6, 27K protein moves slightly faster than TBG. The results of equilibrium sedimentation indicate a mol wt of 66,000, suggesting that the 27K protein might exist as a dimer. These data indicate that the 27K protein is a previously unrecognized T4-binding protein with a low affinity for the hormone. Further studies are required to clarify its physiological role in the transport of circulating thyroid hormones.

[Polymorphism of human thyroxine binding serum globulin (TBG); existence of a new form of serum protein without detectable triiodothyronine binding power]. / Polymorphisme de la globuline du sérum humain fixant la thyroxine TBG; existence d'une forme nouvelle de protéine sérique sans pouvoir fixateur décelable de triiodothyronine.

Two kinds of TBG polymorphism are described in human, one found in deglycosylated TBG from individual blood donors, the other is a genetically determined polymorphism. TBG from plasma samples from a patient with toxic goiter, not autoimmune, (p)TBG, from the patient's mother (m)TBG and from individual donors (n)TBG, were labeled with [125I]T4 or [125I]T3 and submitted to isoelectric focusing (IEF), followed by autoradiography. Three faint [125I]T4 radiolabeled bands were detectable in (p)TBG while four strong [125I]T4 radiolabeled bands were detectable in (m)TBG and (n)TBG), respectively. IEF of the [125I]T3 incubated serum samples resulted in no detectable isoelectric radiolabeled band for (p)TBG while a normal pattern was found in (m)TBG and in (n)TBG, respectively. These data suggest a new intraindividual not linked to sexual chromosome X polymorphism characterized by a loss in hormone binding.

[Effects of enzymatic deglycosylation of human goiter thyroglobulin on its immunochemical properties]. / Les effets de la déglycosylation enzymatique de la thyroglobuline du goitre humain sur ses propriétés immunochimiques.

Thyroglobulin (Tg), isolated from soluble iodoproteins by ammonium sulphate fractionation, was enzymatically deglycosylated in vitro and analyzed by polyacrylamide gel electrophoresis, double immunodiffusion and non-commercial RIA. Carbohydrate and iodine content was chemically determined. By PAAGE deglycosylated Tg (dTg) showed the appearance of a major band in the 12S region and three slower migrating bands corresponding to higher aggregates than 19S Tg. In immunodiffusion by testing native and deglycosylated Tg against anti-native Tg antiserum it was shown the appearance of a spur of native on deglycosylated Tg. By RIA of native and deglycosylated Tg against anti-deglycosylated Tg antiserum it was shown a minor binding capacity of the anti-deglycosylated antibody against native Tg at high dilutions. The results demonstrate that the enzymatic deglycosylation release almost all the carbohydrates of goiter Tg and that the removal of the carbohydrates of Tg produces a loss of antigenic determinants of the molecule.

Effects of visual angle on perspective reversal for ambiguous patterns.

Reversal rates of an ambiguous figure (the Necker cube) were studied for different pattern sizes covering a range of visual angles theta from approximately 1 to 62 deg. A large number of reversals was obtained for each observer and each pattern in order to examine the statistical distributions of reversal times. A pronounced flattening of the statistical distributions (represented throughout by a gamma distribution) and a growth of the mean duration of each percept, with increasing pattern size was found. A plateau in the range of theta between 5 and 20-30 deg was observed. For larger values of theta two kinds of observers have been identified: for 'fast' observers the inversion rate is little affected by theta, whilst for 'slow' observers, the mean reversal time increases strongly with theta. A tentative model, based on three different contributions to the duration of the alternation process, is proposed: a constant term, independent of theta, and two terms dependent on theta--a retinal term, and a cortical one. The last term is interpreted as due to the spreading of excitation with the characteristic of a filling-in process.

[Tunicamycin inhibition of carbohydrate incorporation into thyroglobulin]. / Inhibition par la tunicamycine de l'incorporation des glucides dans la thyroglobuline.

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.

[Effect of change of habitat (sea and fresh water) on in vivo thyroglobulin synthesis in Atlantic glassed eels (Anguilla anguilla L.)]. / Influence du changement d'habitat (eau de mer et eau douce) sur la biosynthèse de la thyroglobuline in vivo chez des civelles d'anguilles atlantiques, Anguilla anguilla L.

Thyroid biosynthesis in glassed eels (Anguilla anguilla L.) was studied to establish whether salinity changes could affect it, when they live in sea water or in fresh water containing 125I. Aqueous extrait of homogenized cephalic heads of glassed eels contains an iodinated protein 17-19 S having thyroglobulin-like properties and including iodotyrosins (MIT and DIT) and thyroid hormones (3 and T4). Biosynthesis of this proteins is roughly twice more important in fresh water than in sea water at 19-21 degrees C and its specific radioactivity (125I) is practically double in fresh water.