A humanised anti-IGF-1R monoclonal antibody (AVE1642) enhances Bortezomib-induced apoptosis in myeloma cells lacking CD45.

The humanised form of an antagonistic anti-IGF-1R mAb (AVE1642) selectively inhibits the growth of CD45(neg) myeloma cells. AVE1642 strongly increased bortezomib-induced apoptosis, correlated with an increase of Noxa expression. These results support the therapeutic use of anti-IGF-1R/bortezomib in CD45(neg) Myeloma patients, particularly those with the most aggressive form, t(4,14).

Reactivation of human herpesvirus type 6 in multiple organ failure syndrome.

Human herpesvirus type 6 (HHV-6) and cytomegalovirus (CMV) are known to interact with the production of cytokines. In this study, we sought to determine the incidence of HHV-6 and CMV reactivation during multiple organ failure syndrome (MOFS) and to evaluate the potential effects of viral replication on both the morbidity and mortality associated with MOFS. Viral reactivation was assessed by use of specific polymerase chain reaction (PCR) analysis of the serum samples obtained from 48 consecutive patients with MOFS (the MOFS group) and from 48 sex- and age-matched patients with <2 organ failures (the control group). In addition, HHV-6 replication was assessed in 106 blood donors (the normal group). The incidence of HHV-6 replication was higher in the MOFS group than in the control and normal groups (26 [54%] of 48 vs. 7 [15%] of 48 and 5 [5%] of 106, respectively; P<.0001), with apparently no influence on morbidity and mortality rates. In contrast, reactivation of CMV was found in a single patient. Further studies are needed to evaluate the pathogenesis of HHV-6 replication in critically ill patients.

Upregulation of the apoptosis-associated protein Grb3-3 in HIV-1-infected human CD4(+) lymphocytes.

The mechanism(s) by which HIV-1 infection contributes to depletion of CD4(+) T cell is not well understood. In this report, we investigated whether a recently identified isoform of growth factor receptor bound protein (Grb2), named Grb3-3, a signaling molecule that is associated with the MAP kinase pathway and with apoptosis could be involved. We find that Grb3-3 is markedly up-regulated following HIV-1 infection of CD4(+) peripheral blood mononuclear cells undergoing apoptosis. Although IL-2 deprived CD4(+) cells also undergo apoptosis to a similar extent, Grb3-3 upregulation is not detected under these experimental conditions. Transient overexpression of Grb3-3 in Jurkat T-cells also causes apoptosis. Upon staurosporine stimulation, Grb3-3 predisposes Sup-T1 cell to apoptosis. Finally, analysis of the HIV-1 genes responsible for Grb3-3 expression demonstrates that Tat and Nef can independently induces its expression, suggesting these two earliest viral gene products of HIV-1 may share some common pathway(s) in up-regulating Grb3-3 expression.

Grb3-3 is up-regulated in HIV-1-infected T-cells and can potentiate cell activation through NFATc.

The MAPK pathway is required for T-cell activation; however, its role in modulating T-cell function following human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. In this report, we investigated whether Grb3-3, an isoform of the Grb2 (growth factor receptor-bound protein-2) adaptor molecule that is associated with the MAPK pathway, could be involved. We found that Grb3-3, but not its isoform Grb2, is markedly up-regulated in CD4(+) peripheral blood mononuclear cells derived from either in vitro HIV-1-infected cultures or HIV-1-infected human subjects. Analysis of HIV-1 gene products indicated that Tat and Nef, both of which have been implicated in modulating T-cell function, can independently induce expression of Grb3-3. By using NFAT/AP-1, AP-1, or NFAT reporter assays, we found that Grb3-3 can potentiate NFAT (but not AP-1) promoter activity in Jurkat T-cells upon engagement of the T-cell receptor and CD28 co-receptor. In addition, potentiation of NFAT by Grb3-3 is substantially suppressed by MEKK1, a kinase that may play an important role in retaining NFAT in the cytoplasm, and by cyclosporin A. Finally, we also found that Grb3-3 potentiates HIV-1 long terminal (LTR) repeat promoter activity following T-cell receptor stimulation, an effect that can be largely suppressed by cyclosporin A. Taken together, this study indicates that Grb3-3 is a cellular factor that can be up-regulated by HIV-1. In addition, Grb3-3 can also function as a positive factor for T-cell activation and, in doing so, may aid in establishing an intracellular environment that can optimally support HIV-1 replication.

[Ocular bacterial infections : present and prospective methods of diagnosis]. / Infections oculaires bactériennes: méthodes di diagnostic actuelles et prospectives.

Bacteriological samples and tests are essentiel for the diagnosis of superficial ocular infections and endophtalmitis. The direct examination and the traditional culture of the samples can be in the futur associated with new diagnostic approach using antigen detection (immunofluorescence, enzyme immunosorbent assays) and genome research by hybridation or better by amplification for Chlamydia and for the most frequent species responsible of endophtalmitis. An original genomic strategy of bacterial endophtalmitis diagnosis was developped.

Evaluation of BC-3 and BCP-1 cell lines in immunofluorescence assay to detect HHV-8 antibodies in Kaposi's sarcoma, multiple myeloma and other groups.

BACKGROUND: We describe a comparative study of an immunofluorescence assay using inducible BC-3 and BCP-1 cell lines as sources of HHV-8 antigens. STUDY DESIGN: Detection of both antibodies to proteins expressed in lytic cycle and during latency in sera from HIV-infected patients with KS, HIV-positive patients without KS, normal blood donors, HIV-negative pregnant women and HIV-negative patients with multiple myeloma. Where possible, detection of antibody was associated with nested PCR detection of HHV-8 in peripheral mononuclear cell (PBMC) samples collected from AIDS-KS patients. RESULTS: Immunofluorescence was more intense with the BC-3 cell line than with BCP-1, thus facilitating examination under the microscope. HHV-8 antibodies were detected among 82.75% of AIDS-KS patients, in 27.3% of HIV-infected homosexual men, 2% of blood donors and in 2% of pregnant women. No HHV-8 antibodies were detected in serum samples from HIV-negative patients presenting multiple myeloma. HHV-8 DNA sequences were detected and confirmed by southern blot hybridization in five out of 17 (29.4%) PBMC samples from AIDS-KS patients. Titre of antibodies to proteins expressed in lytic cycle was much higher than the titre of antibodies to proteins expressed during latency. CONCLUSIONS: Both immunofluorescence assays were found useful and HHV-8 seroprevalence rates reported in previous studies were confirmed. In addition, results obtained using these assays tend to provide evidence for a lack of epidemiological association between HHV-8 infection and development of multiple myeloma.

Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains.

The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.g. BAX and PICG3. Additionally, p53 can repress a variety of promoters, which, in turn, may be responsible for the functional activities exhibited by p53. This study shows that the Q22, S23 double mutation, which is known to inactivate a p53 transactivation subdomain located within the initial 40 residues of the protein, while abrogating transactivation from the WAF1 promoter, only attenuates apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. The Q53, S54 double mutation, which inactivates another p53 transactivation subdomain situated between amino acids 43 and 73 results in attenuation of all of the aforementioned p53 activities. In contrast to the Q22, S23 double mutation, this latter mutation set does not alter mdm-2-mediated inhibition and degradation of p53. Finally, mutation of all four residues results in complete abrogation of every p53 activity mentioned above.

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments.

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

A tumor specific single chain antibody dependent gene expression system.

The design of conditional gene expression systems restricted to given tissues or cellular types is an important issue of gene therapy. Systems based on the targeting of molecules characteristic of the pathological state of tissues would be of interest. We have developed a synthetic transcription factor by fusing a single chain antibody (scFv) directed against p53 with the bacterial tetracycline repressor as a DNA binding domain. This hybrid protein binds to p53 and can interact with a synthetic promoter containing tetracycline-operator sequences. Gene expression can now be specifically achieved in tumor cells harboring an endogenous mutant p53 but not in a wild-type p53 containing tumor cell line or in a non-transformed cell line. Thus, a functional transactivator centered on single chain antibodies can be expressed intracellularly and induce gene expression in a scFv-mediated specific manner. This novel class of transcriptional transactivators could be referred as 'trabodies' for transcription-activating-antibodies. The trabodies technology could be useful to any cell type in which a disease related protein could be the target of specific antibodies.

[Human herpesviruses 6 and 7 (HHV-6 and HHV-7)]. / Herpesvirus humains 6 et 7 (HHV-6 et HHV-7).

Human herpesviruses 6 (HHV-6) and 7 (HHV-7) are world wide T lymphotropic viruses recently discovered. Their transmission is essentially by salivary route. Primary infections which occurred early in infancy, respectively during the first year of life for HHV-6 and in the second or third year for HHV-7, are followed by latency for life. HHV-7 is not actually associated with a disease. HHV-6 primary infection is often asymptomatic, if not it can induce exanthem subitum. HHV-6 reactivation can be symptomatic in immunodeficient subjects. The role of HHV-6 in the arising of lymphoproliferative or auto-immune diseases, discussed for a long time, is still to elucidate. HHV-6 infection is diagnosed by serodiagnosis in case of primary infection, but in the great number of cases, it would be realized by polymerases chain reaction.

Genital recurrent infection occurring 6 months after meningitis due to the same herpes simplex virus type 2 (HSV-2) strain evidence by restriction endonuclease analysis.

Herpes simplex virus type 2 (HSV-2) is more often sexually transmitted and associated with genital recurrent infection. However, HSV-2 neurological manifestations such as meningitis were already reported. We describe a case of meningitis due to HSV-2, preceded by signs suggesting a common cystitis, in a woman with no history of primary or recurrent genital infection. Six months later genital herpetic lesions occurred. One HSV-2 strain was obtained from cerebrospinal fluid (CSF) and another from genital lesions. The molecular comparative analysis using restriction endonuclease digestion patterns showed the similarity of the two strains. Our report illustrates that HSV-2 infections are underdiagnosed and that molecular techniques can be of value in clarifying the physiopathology of HSV diseases.

Grb2 interaction with MEK-kinase 1 is involved in regulation of Jun-kinase activities in response to epidermal growth factor.

Epidermal growth factor (EGF) receptor was shown to be involved in the activation pathway of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) cascade not only by EGF, but also by UV radiation or osmotic stress. This paper describes a specific interaction between the COOH-terminal SH3 domain of Grb2 and the NH2-terminal regulatory domain of MEKK1 in ER22 cells overexpressing the EGF receptor. This interaction results in the formation of a constitutive complex between Grb2 and MEKK1 in both proliferating and resting cells. EGF stimulation causes this complex to be rapidly and transiently recruited by Shc proteins. The subsequent release of the Grb2-MEKK1 complex from Shc proteins correlates with JNK activation. Transfection of the NH2-terminal regulatory domain of MEKK1 specifically inhibits EGF-dependent JNK activation indicating that Grb2 is involved in MEKK1 activation. Thus, adaptor proteins have a new role in the regulation of the SAPK/JNK cascade after EGF stimulation.

The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression.

Wild-type p53 is a tumor suppressor gene which can activate or repress transcription, as well as induce apoptosis. The human p53 proline-rich domain localized between amino acids 64 and 92 has been reported to be necessary for efficient growth suppression. This study shows that this property mainly results from impaired apoptotic activity. Although deletion of the proline-rich domain does not affect transactivation of several promoters, such as WAF1, MDM2 and BAX, it does alter transcriptional repression, reactive oxygen species production and sequence-specific transactivation of the PIG3 gene, and these are activities which affect apoptosis. Whereas gel retardation assays revealed that this domain did not alter in vitro the specific binding to the p53-responsive element of PIG3, this domain plays a critical role in transactivation from a synthetic promoter containing this element. To explain this discrepancy, evidence is given for a proline-rich domain-mediated cellular activation of p53 DNA binding.

CTS1: a p53-derived chimeric tumor suppressor gene with enhanced in vitro apoptotic properties.

The clinical potential of the p53 tumor suppressor gene is being evaluated currently for gene therapy of cancer. We have built a variant of wild-type p53, chimeric tumor suppressor 1 (CTS1), in which we have replaced the domains that mediate its inactivation. CTS1 presents some very interesting properties: (a) enhanced transcriptional activity; (b) resistance to the inactivation by oncogenic forms of p53; (c) resistance to the inactivation by MDM2; (d) lower sensitivity to E6-induced degradation; (e) ability to suppress cell growth; and (f ) faster induction of apoptosis. Thus, CTS1 is an improved tumor suppressor and an alternative for the treatment of wild-type p53-resistant human tumors by gene therapy.

[Rhinovirus infections in hospitalized children: a 3-year study]. / Infections à rhinovirus chez des enfants hospitalisés: étude sur une période de 3 ans.

BACKGROUND: Rhinoviruses (RH) are responsible for acute respiratory illnesses, mainly in the upper respiratory tract. POPULATION AND METHODS: 3,152 children aged under 16 years, admitted to the Paediatrics department of the University Hospital Centre of Poitiers from January 1, 1993 to December 31, 1995 with ear, nose and throat (ENT) and/or respiratory symptoms were systematically investigated. One hundred and forty-five RH strains were isolated from nasopharyngeal secretions of 87 boys and 58 girls (mean age: 20.3 months). Among these, 92 (63.4%) were less than 1 year of age. Bacteriological investigations were done for 29 patients when a concomitant bacterial infection was suspected. RESULTS: RH infection rate was maximum before 1 year of age (median age: 6.5 months) and decreased with age. RH were isolated throughout the 3 years, with a first peak from February to April, and a second one in autumn. The main symptoms were sibilants (27.6%) and cough (24.1%). Sibilants were more frequently associated in children under 12 months of age (P = 0.01). Sometimes, ophthalmologic or digestive symptoms were present. Three children with respiratory distress were transferred to the reanimation ward. In addition, a RH strain was isolated from a child who died of sudden infant death. Thirty-four children (23.4%) were co-infected by one or several viruses; the most frequently detected were the respiratory syncytial virus (41.2%) and the adenoviruses (35.3%). Twenty-nine children were infected by two viruses and five by three. Associated bacterial infections were diagnosed in 23 children, especially conjunctivitis due to Haemophilus influenzae (21.7%). Among these children, eight had a multiple viral infection. CONCLUSION: RH have a limited pathogenicity but can be associated with serious illnesses among infants and children.

[Virus and gastrointestinal infections]. / Virus et infections gastro-intestinales.

Numerous viruses found in the gut are not associated with primary infection or disease of the gastrointestinal tract. Other groups or viruses are not classically associated with infection of the gut but can infect the gastrointestinal tract in immunocompromised individuals (herpes simplex virus, cytomegalovirus, papillomavirus ....). The viruses associated with gastroenteritis represent a large number of taxonomic group. Because these viruses have in general been difficult to cultivate, most members of this group were firstly detected by electron microscopic examination (adenovirus, astrovirus, calicivirus, coronavirus, rotavirus ....). The most widely used diagnostic techniques for adenovirus (40/41), rotavirus and astrovirus detection in faecal samples include immunoassays such as Elisa and latex agglutination. Nucleic acid hybridization techniques have generally not proven to be substantially sensitive and the more sensitive techniques recently developed use the polymerase chain reaction (adenovirus) or the reverse transcription/polymerase chain reaction (astrovirus, calicivirus, coronavirus, rotavirus). Special efforts have been made in the search for efficient procedures to extract viral nucleic acids, and to establish the optimal conditions for the amplification and identification of PCR products but the candidate viruses were very different, consensus procedures were not determined, and amplification kits were not actually commercialized.

[Action of isoconazole on Candida albicans. Study by scanning and transmission electron microscopy]. / Action de l'isoconazole sur Candida albicans. Etude en microscopie électronique à balayage et à transmission.

Candida albicans yeast cells were treated prior to their growth phase with isoconazole at the following concentrations: 0.1 microgram/ml, 1 microgram/ml, 10 micrograms/ml), isoconazole induces a blockade of cell diprecipitates bearing witness to the presence of polysaccharides were noted in the cytoplasma and against the membrane wall, but cell division was apparently unaffected. At low concentrations (1 microgram/ml, 10 microgram/ml), isoconazole induced a blockade of cell division by its fungicidal action on synthesis and organisation of the membrane wall. At higher concentrations (50 microgram/ml; 100 micrograms/ml), isoconazole induces total necrosis and death. These findings confirm the analogy between the fungicidal actions of isoconazole and miconazole; however, the site of action of imidazole derivatives (wall or plasma membrane) is discussed.

Ultrastructural study of Candida albicans yeast after application of a ribonuclease.

Electron microscopy was performed on Candida albicans yeast after application of ribonuclease to clear the cytoplasmic background. In conjunction with Thiery's method of highlighting polysaccharide components, this clearing technique, which has not been used since 1959, enabled visualization of the nucleus, the mitochondria, the vacuolar system and another structure which seemed to be the Golgi apparatus. The invaginations of the plasmalemma membrane (or lomasomes) are highly developed and may be partially responsible for transporting material required for development of the cell wall, especially during budding.