Molecular arrangement between multivalent glycocluster and Pseudomonas aeruginosa LecA (PA-IL) by atomic force microscopy: influence of the glycocluster concentration.

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA-IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA-IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra-galactosylated glycocluster and the PA-IL lectin for high concentration of lectins (2.5 µM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one-dimensional elongated aggregates to two-dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction.

Evidence for autotetraploidy associated with reproductive isolation in Saccharomyces cerevisiae: towards a new domesticated species.

Partial or whole-genome duplications have played a major role in the evolution of new species. We have investigated the variation of ploidy level in a panel of domesticated strains of Saccharomyces cerevisiae coming from different geographical origins. Segregation studies and crosses with tester strains of different ploidy levels showed that part of the strains were well-balanced autotetraploids displaying tetrasomic inheritance. The presence of up to four different alleles for various loci is consistent with a polyploidization mechanism relying on the fusion of two nonreduced meiospores coming from two S. cerevisiae strains. Autotetraploidy was also in accordance with karyotype and flow cytometry analyses. Interestingly, most bakery strains were tetraploids, suggesting a link between ploidy level and human use. The null or drastically reduced fertility of the hybrids between tetraploid and diploid strains indicated that domesticated S. cerevisiae strains are composed of two groups isolated by post-zygotic reproductive barriers.

[End of the word and relegation of bodies in medicine: university formation of physicians]. / La fin de la parole et la relégation du corps en médecine: ses conséquences sur la formation Universitaire des Médecins.

Medicine is based on a growing demand for science. Yet, the patient's speech is ill adapted to the current desire of rationality. Inaccuracy and errancy have become the features of clinical examination. Self censorship of the speech gradually appears. Presenting the body becomes useless, if not suspicious. Medical technology replaces perceived subjectivity. What medicine says prevails over what the body knows. In a strange paradox, the "echo" precedes the speech. The risk of having an autistic medicine is looming. University teaching must be aware of this. The obvious gap between the speech and the body not only evades the ethical issue but also paradoxically deprives the medicine from its scientific acumen.

Impairment of the growth of Plasmodium falciparum in HbEE erythrocytes.

HbE is a beta-chain mutant frequently found among inhabitants of Southeast Asia and surrounding territories. We find that Plasmodium falciparum multiplies more slowly in erythrocytes from individuals homozygous for HbE than in cells from HbA individuals. In contrast, this parasite grows normally in erythrocytes heterozygous for HbE. This is the first direct evidence that suggests what has been suspected on the basis of circumstantial data, that HbE-containing erythrocytes might be advantageous to the carrier in regions with endemic malaria.

[Blood transfusion and ethics: new questions]. / Ethique et transfusion: reposer les questions.

Chairman to the French Institutional Review Board, Professor Didier Sicard raises blood donation issues from an ethical standpoint. The contaminated blood scandal focused on the necessity of reducing transfusion risks and regarded blood safety as an ethical mandatory requirement, a debatable subject to deal with. The author proposes to reconsider the nature of unpaid blood donations while advising not to scorn the remunerated gift when such is the case. As for the use of blood, he questions the solutions based on a zero risk perspective, in particular an excessive auto-transfusional practice or a restrictive use of blood, lately regarded as essential. Starting from the blood donation concern this article leads us to think over both our society's fears and the precautionary principle abuses.

[Whipple disease, initially diagnosed as sarcoidosis]. / Maladie de Whipple diagnostiquée primitivement sarcoïdose.

INTRODUCTION: Whipple disease is a multisystem infectious disease caused by Tropheryma whipplei. We report a case in which an initial diagnosis of sarcoidosis was changed to Whipple disease endocarditis. CASE: Based on clinical, radiographic, endoscopic and histologic findings, this 61-year-old man was diagnosed with sarcoidosis. Initial response to corticotherapy was good, but the patient required 35 mg of prednisone daily. The subsequent onset of clinical and laboratory signs of inflammation cast doubt on the diagnosis. After cardiac ultrasound revealed a mass 1 cm in diameter on the mitral valve, apparently vegetation, we diagnosed culture-negative infective endocarditis and ruled out most possible causes. PCR of a duodenal biopsy sample showed Tropheryma whipplei, thus confirming the diagnosis of Whipple disease, despite normal histological findings. After 3 weeks of intravenous gentamicin and amoxicillin treatment, oral cotrimoxazole was substituted. Follow-up transesophageal ultrasound showed no mitral vegetation. The patient, still under cotrimoxazole, has been off prednisone for 13 months and is completely asymptomatic. CONCLUSION: This case is an illustration of the difficulty in distinguishing Whipple disease from sarcoidosis in practice and of the importance of that distinction.

[Prevalence and risk factors of hepatitis A infection in an HIV-infected French population]. / Prévalence et facteurs de risque de l'hépatite A au sein d'une population de patients infectés par le virus de l'immunodéficience humaine.

BACKGROUND: There are common risk factors between hepatitis A virus (HAV) and human immuno deficiency virus (HIV) infections. OBJECTIVES: We tried to evaluate if HIV-infected patients could be at risk for HAV. More over, HAV could worsen prognosis of HIV infection and HAV vaccination was then to be considered. Thus we assessed the prevalence and risk factors of HAV infection in an HIV-infected population. PATIENTS AND METHODS: Seroprevalence and risk factors for HAV were studied among 154 HIV-positive patients followed in a Parisian hospital (mean age: 42 years, male patients: 70.8%, female patients: 29.2%). They were screened for HAV antibodies and answered a questionnaire on risk factors for HAV and means of HIV contamination. RESULTS: The global prevalence was 72.7% [IC95%: 65.7-79.7]. We excluded patients who were born in highly endemic areas where seroprevalence reached 60% [IC95%: 51.2-70]. The HAV seroprevalence was almost 100% in migrants from highly endemic countries and for those born before 1946. The multivariate analysis showed that risk factors were the geographic origin [OR=20.88; IC95%: 2.40-181], age [OR = 2.33; IC95%: 1.24-4.39], and hemophilia [OR = 13.78; IC95%: 1.34-141]. CONCLUSION: Our results suggest that a screening test for HAV antibodies should be performed before vaccination, especially in HIV-infected patients born after 1946 or in non-endemic countries.

Qualitative and quantitative analysis of human cytotoxic T-lymphocyte responses to HIV-1 proteins.

OBJECTIVE: To study the degree of immunogenicity of each HIV-1 protein. DESIGN: In most viral systems, antiviral cytotoxic T-lymphocytes (CTL) from a given donor preferentially recognize only one or a small number of viral proteins. METHODS: Anti-HIV CTL were generated by in vitro stimulation of peripheral blood mononuclear cells from seropositive donors and tested against multiple HIV-1 proteins or groups of proteins encoded by seven genes (env, gag, pol, nef, rev, tat and vif). Using autologous target cells infected with recombinant vaccinia viruses expressing one of the HIV-1LAI proteins, we compared the cytolytic activities obtained from bulk culture with those found in limiting dilution analysis (LDA). RESULTS: Our results were noteworthy for the following reasons. (1) Each responding donor reacted simultaneously to multiple proteins; this is very unusual in other viral systems. Anti-Gag CTL were detected in most, and anti-Pol in approximately three-quarters, of the patients, together with very high amounts of the corresponding CTL precursors in LDA. CTL against Env and Nef were found in two-thirds of the patients, while Vif- and Rev-specific CTL were less frequent. Finally, Tat was seldom recognized by CTL, but its antigenicity was revealed in LDA. (2) All responding cells revealed in bulk cultures as well as in LDA were CD8+ T-cells, and their in vitro differentiation did not require the help of CD4+ T-cells. (3) Proteins from the HIV-1LAI isolate were recognized with high frequency by CTL from seropositive donors, most certainly being infected by other isolates, which suggests that relatively conserved epitopes are predominant targets of CTL. CONCLUSION: Taken together, these results are encouraging for vaccine purposes, since anti-HIV-1 CTL stimulation is thought to be a requirement for such a vaccine.

Detection and quantification of HIV-1 in semen: identification of a subpopulation of men at high potential risk of viral sexual transmission.

BACKGROUND: To assess HIV burden in both acellular and cellular fractions of semen in men with different levels of blood plasma HIV RNA by a cross-sectional study. PATIENTS: Fifty-two HIV-1-seropositive men (21 receiving antiretroviral therapy) with CD4 cell counts ranging from 1 to 1170 x 10(6)/l. METHODS: Semen was separated into seminal plasma and fractions enriched in motile spermatozoa or non-spermatozoal cells. HIV RNA was quantified by the HIV-Monitor technique (Roche) in blood plasma, seminal plasma and spermatozoa fractions. HIV DNA or infectious virions in cellular fractions were detected by either PCR or qualitative viral culture. RESULTS: HIV RNA was detected in 86.5% of seminal plasma specimens and in 14.6% of spermatozoa fractions; HIV DNA was detected in 57.1% of non-spermatozoal cell fractions. HIV RNA levels in blood plasma and seminal plasma were correlated (r5 = 0.56, P < 0.0001, Spearman's rank test). A majority of men had lower levels in seminal plasma than in blood plasma: one-third had HIV-positive seminal cell fractions. However, 20 men (38.5%) with HIV RNA levels in seminal plasma (median: 4.65 log10 copies/ml) comparable to or higher than those in blood plasma had all HIV-positive non-spermatozoal cells or spermatozoa fractions with a high frequency of positive cultures. CONCLUSION: A high frequency of men had detectable HIV in semen. We identified a subpopulation demonstrating high levels of HIV RNA in seminal plasma, comparable to or higher than those in blood plasma, frequently associated with a substantial viral shedding in seminal cells, raising the possibility of viral production within the genital tract and suggesting heterogeneity in the potential of HIV sexual transmission among infected men.

Identification of an ancestral resistance gene cluster involved in the coevolution process between Phaseolus vulgaris and its fungal pathogen Colletotrichum lindemuthianum.

The recent cloning of plant resistance (R) genes and the sequencing of resistance gene clusters have shed light on the molecular evolution of R genes. However, up to now, no attempt has been made to correlate this molecular evolution with the host-pathogen coevolution process at the population level. Cross-inoculations were carried out between 26 strains of the fungal pathogen Colletotrichum lindemuthianum and 48 Phaseolus vulgaris plants collected in the three centers of diversity of the host species. A high level of diversity for resistance against the pathogen was revealed. Most of the resistance specificities were overcome in sympatric situations, indicating an adaptation of the pathogen to the local host. In contrast, plants were generally resistant to allopatric strains, suggesting that R genes that were efficient against exotic strains but had been overcome locally were maintained in the plant genome. These results indicated that coevolution processes between the two protagonists led to a differentiation for resistance in the three centers of diversity of the host. To improve our understanding of the molecular evolution of these different specificities, a recombinant inbred (RI) population derived from two representative genotypes of the Andean (JaloEEP558) and Mesoamerican (BAT93) gene pools was used to map anthracnose specificities. A gene cluster comprising both Andean (Co-y; Co-z) and Mesoamerican (Co-9) host resistance specificities was identified, suggesting that this locus existed prior to the separation of the two major gene pools of P. vulgaris. Molecular analysis revealed a high level of complexity at this locus. It harbors 11 restriction fragment length polymorphisms when R gene analog (RGA) clones are used. The relationship between the coevolution process and diversification of resistance specificities at resistance gene clusters is discussed.

Tuberculosis due to Mycobacterium bovis after alemtuzumab administration.

We describe a patient with relapsing B chronic lymphocytic leukemia who developed systemic bacille Calmette-Guérin infection (BCGitis) after administration of alemtuzumab (Campath-1H).

Development and standardization of methods to evaluate the antibody response to an HIV-1 candidate vaccine in secretions and sera of seronegative vaccine recipients.

Enzyme-linked immunosorbent assays (ELISA) were developed to test, in serum and mucosal samples, total IgG, total IgA, serum albumin, and anti-gp120 MN and anti-p24 LAI IgG and IgA levels. These ELISAs were optimized according to reagents and experimental conditions. Inter- and intra-assay coefficients of variation ranged from 3.3% to 18.6%. The ELISA results were linear and precise, and for anti-HIV-1 IgG and IgA, the analytical recovery was close to 100%. For IgG and IgA titration against gp120 MN and p24 LAI, standards were made using pooled sera or gammaglobulins with assigned titres in ELISA units per ml (EU/ml). These standards were used to obtain a linear regression curve that could then be used to obtain the titres of experimental samples. The cut-offs for positivity were determined for sera and mucosal fluid using healthy controls. Validation conditions were defined for ELISAs, and samples that did not satisfy these conditions were retested. Measurement of total IgG and IgA allowed normalization and comparison of the results of specific immunoglobulin levels between different samples. Serum albumin was tested as a marker of transudation from serum to mucosal fluid, allowing calculation of the relative coefficient of excretion, which is one element required to determine the origin of the immunoglobulin detected in mucosal samples. These ELISAs were developed with samples from HIV-1-infected and healthy subjects. We now have the tools to study and understand mucosal immunity in seronegative subjects vaccinated with an HIV-1 candidate vaccine.

Acquired hemophilia due to factor VIII inhibitors in 34 patients.

BACKGROUND: Acquired hemophilia is a rare disease caused by the development of auto-antibodies against factor VIII. SUBJECTS AND METHODS: We studied the characteristics and outcomes of 34 patients (19 women and 15 men) with acquired hemophilia from 1980 to 1997. RESULTS: The mean age of the patients was 61 years (range, 22-93 years). An underlying disease was observed in 18 (53%) patients: 5 patients had cancer, 4 an autoimmune disorder, 2 a dermatologic disorder, 3 asthma, 3 were postpartum, and 1 had an adverse reaction to ampicillin. Factor VIII level was <5% in 30 (90%) patients; factor VIII antibodies were elevated (>10 Bethesda units) in 23 (69%) patients. Bleeding requiring transfusions was reported in 25 (75%) patients. Human factor VIII was given to 14 patients and porcine factor VIII to 5. Six patients received prothrombin complex concentrates and one desmopressin. Several immunosuppressive treatments were used, mainly corticosteroids, cyclophosphamide, and intravenous immunoglobulin. Bleeding stopped in all but one patient within 2 weeks. Most patients achieved complete remission, although two relapses were observed subsequently. CONCLUSION: This large study helps to clarify the presentation and clinical course of acquired hemophilia. Prospective studies are needed to determine the efficacy of treatment.

Heparin cofactor II deficiency in patients infected with the human immunodeficiency virus.

In human plasma, heparin cofactor II (HCII) is a thrombin inhibitor, whose deficiency has been reported to be associated with recurrent thrombosis. The finding of two cases of low plasma HCII activity in two patients infected with the human immunodeficiency virus (HIV) led us to investigate this coagulation inhibitor in the plasma of a larger population of HIV-infected patients. The mean plasma HCII activity was significantly lower in 96 HIV-infected patients than in 96 age- and sex-matched healthy individuals (0.75 +/- 0.24 vs 0.99 +/- 0.17 U/ml, p < 0.0001). HCII antigen concentration was decreased to the same extent as the activity. The proportion of subjects with HCII deficiency was significantly higher in the HIV-infected group than in healthy individuals (38.5% vs 2.1%). In addition, HCII was significantly lower in AIDS patients than in other HIV-infected patients, classified according to the Centers for Disease Control (CDC) on the basis of an absolute number of circulating CD4+ lymphocytes below 200 x 10(6)/l. The link between HCII and immunodeficiency is further suggested by significant correlations between HCII activity and both the absolute number of CD4+ lymphocytes and the CD4+ to CD8+ lymphocyte ratio. Nevertheless, the mean HCII level was not different in the various groups of patients classified according to clinical criteria, except in CDC IVD patients in whom HCII levels were significantly lower. In addition, no correlation could be demonstrated between HCII and protein S activities, another coagulation inhibitor whose plasma level was also found to be decreased in HIV-infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrin polymerization defect in HIV-infected patients--evidence for a critical role of albumin in the prolongation of thrombin and reptilase clotting times.

Thrombin clotting time (TCT) and reptilase clotting time (RCT) were found significantly prolonged in a series of 72 HIV-infected patients drawn for routine coagulation testing. Both TCT and RCT were highly significantly correlated with albumin (r = -0.64, and r = -0.73 respectively, p < 0.0001). TCT and RCT were significantly higher (p < 0.0001) in a series of 30 other HIV-infected patients selected on their albumin level below 30.0 g/l (group 1) than in 30 HIV-infected patients with albumin level above 40.0 g/l or in 30 HIV-negative controls; the two latter groups were not different. In vitro supplementation of plasma from group 1 patients with purified human albumin up to 45.0 g/l (final concentration) lead to a dramatic shortening effect on both TCT and RCT, which reached normal values. The TCT and RCT of the purified fibrinogen solutions (2.0 g/l final concentration) were not different in the three groups, and normal polymerization curves were obtained in all cases. This further ruled out the presence of any dysfibrinogenemia in the plasma from group 1 patients. Using purified proteins, highly significant correlations were demonstrated between the albumin concentration and the prolongations of both TCT and RCT, which were of the same magnitude order than those found in the patients plasma. These results suggest that hypoalbuminemia is responsible for the acquired fibrin polymerization defect reported in HIV-infected patients. The pathophysiological defect reported in HIV-infected patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed virus-specific CD8+ cytotoxic T lymphocyte activity in an HIV-infected individual with high CD4+ cell counts: correlations with various parameters of disease progression.

This 4-year longitudinal study monitored the temporal association between the HIV-specific cytotoxic T lymphocyte (CTL) response and the control viremia in an individual infected with human immunodeficiency virus type (HIV-1). At the beginning of the study, this asymptomatic individual with a high CD4+ cell count showed no HIV-specific cytotoxic activity after polyclonal in vitro restimulation with autologous PHA-blasts, unlike most HIV-seropositive individuals. Anti-HIV CTLs were detected only in the last year of the study, both after in vitro restimulation and directly ex vivo. This was correlated with the inversion of the CD4+/CD8+ ratio, essentially due to increased numbers of CD8+CD28- T lymphocytes. The HIV-specific cytolytic activity was mediated by this CD28+CD28- subpopulation. The amount of HIV-1 provirus in peripheral blood mononuclear cells (PBMCs) did not change during the study, but the HIV RNA in plasma increased and virus was isolated from PBMCs only at the time when HIV-specific CTL activity was detected. This suggests overall that the HIV-1 replication was low in this individual, with a transient increase that could have reached the threshold for CTL reactivation, and was perhaps controlled thereby.

HIV type 1-specific IgG2 antibodies: markers of helper T cell type 1 response and prognostic marker of long-term nonprogression.

The helper T type 1 (Th1) function of CD4(+) T lymphocytes is presumed to be of key importance in host defense against HIV-1. As the production of different antibody isotypes is dependent on this helper T function, we investigated whether HIV-1-specific responses of a particular IgG isotype could be a reliable marker of long-term HIV-1 control. Assessment of the IgG subclass distribution in the plasma of HIV-1-infected patients enrolled in the French prospective Asymptomatic Long-Term (ALT) cohort showed that IgG2 directed against HIV-1 Env gp41 and Gag proteins was associated with low viral load, high CD4(+) lymphocyte count, and weak neutralizing activity. By contrast, levels of anti-Env and anti-Pol IgG1 as well as the magnitude of neutralizing activity were correlated with the viral load and thus merely reflect the level of HIV replication. Furthermore, IgG2 directed against Gag proteins was significantly associated with HIV-1 p24-specific Th1 cell production of interferon gamma and interleukin 2. In multivariate analysis, only two variables, anti-gp41 IgG2 and plasma HIV-1 RNA, were found to be independent prognostic factors of remaining long-term nonprogressive over time. By providing new insight into the nature of an HIV-specific antibody response associated with the control of virus replication, these findings have implications for the design of HIV vaccines.

IgG subclass distribution in serum and various mucosal fluids of HIV type 1-infected subjects.

We measured total IgG1, IgG2, IgG3, and IgG4 concentrations by ELISA in serum (S), total saliva (TS), cervicovaginal secretions (CVS), seminal secretions (SPE), and rectal secretions (RS) from either CDC II/III HIV-1-infected subjects or healthy volunteers. Human serum albumin was measured in parallel to calculate the relative coefficient of excretion (RCE). Levels of IgG1 and IgG3 directed against gp120 MN also were measured by ELISA in all samples, and the specific activity (SA) calculated. HIV-1-specific IgG2 and IgG4 were not compared, as total IgG2 and total IgG4 levels in HIV-1-infected subjects were found to be lower than in the healthy controls. Despite substantial interindividual variability, total IgG1 and IgG3 concentrations in all fluids were greater in the HIV-1-infected subjects than in the healthy controls. Calculations of RCE indicated predominantly a transudative origin for IgG subclasses in the different mucosal fluids, except for CVS, in which IgG1, IgG2, and IgG4 was produced locally. The transduction behavior of IgG3 in secretions appears to be different from that of other IgG subclasses. HIV-1-infected subjects were considered positive for IgG1 and IgG3 antibodies against gp120 MN if their antibody levels exceeded the maximum titer measured in the control group. Positive levels of anti-gp120 MN IgG1 were detected for 100% of HIV-1-infected individuals in S, CVS, and SPE, 97% in TS, and 75% in RS. Fewer subjects had positive levels of IgG3 to gp120 MN in their secretions (maximum 67% in CVS). Despite the low concentrations of total IgG3, mean SA values for IgG3 to gp120 MN were greater in secretions than in serum. No significant difference in the SA values for IgG1 to gp120 MN was observed between the different fluids. Only CVS had a local production of HIV-specific IgG1 Our results highlight the importance of an HIV-specific IgG1 and IgG3 immune response in mucosal fluids from HIV-1-infected subjects.