Daniel Drocourt

Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance

Phleomycins are glycopeptide antibiotics related to bleomycins nucleotide sequence of the Sh ble gene and its polylinker and tallysomycins which are active at low concentrations on both fragments. prokaryotic and eukaryotic cells (1). We have cloned a phleomycin-resistance gene (Sh ble) from the genomic DNA of REFERENCES Streptoalloteichus hindustanus (ATCC 31158), a tallysomycin 1 Berdy,J. (1980) Handbook of.Antibiotic Compounds. Vol.4, pp. 459-491, producer. The Sh ble gene encodes a small acidic protein (MW CRC Press, Boca Raton, FL, USA 13665) which has been crystallised for X-ray structural studies 2. Rondeau,J.M., Cagnon,C., Moras,D. and Masson,J.M. (1989) J. MoI. Biol. (2). The Sh protein is a binding protein with a strong affinity 207, 645-646. for the phleomycin family of antibiotics (3). When these 3. Gatignol,A., Durand,H. and Tiraby,G. (1988) FEBS Lent. 230, 171-175. antibiotics are bound by the Sh protein, then phleomycins can 4. Glumoff,V., Kappeli,O., Fiechter,A. and Reiser,J. (1989) Gene 84, nolonger areactivated by tneffous ions and oxyen teobreakdcan 311-318. no longer be activated by ferrous ions and oxygen to break down 5. Mattern,J.E. and Punt,P.J. (1988) Fungal Genet. Newslett. 35, 25. DNA (3). The Sh ble gene has been used in a number of 6. Perez,P., Tiraby,G., Kallerhoff,J. and Perret,J. (1989) Plant Mol. Biol. 13, laboratories as a selectable marker for lower (4, 5) and higher 365-373. eukaryotes (6, 7, 8). In order to facilitate vector construction 7. Mulsant,P., Gatignol,A., Dalens,M. and Tiraby,G. (1988) Somat. Cell. Mol. exeukryoetes(, 8).theinordgoers wto fac tipltevctoricositruction Genet. 14, 243-252. periment , synthetic oligom with multiple cloning sites have 8. Cosset,F.L., Legras,C., Chebloune,Y., Savater,P., Thoraval,P., been added at both ends of the Sh ble gene to give the two pUC Thomas,J.L., Samarut,J., Nigon,U.M. and Verdier,G. (1990) J. Virol. 64, based derivatives, pUT56 and pUT58. We report here the 1070-1078.

Dectin-1 Is Essential for Reverse Transcytosis of Glycosylated SIgA-Antigen Complexes by Intestinal M Cells

This work reports the long-awaited identification of Dectin-1 and Siglec-5 as the M cell co-receptors that mediate the reverse transcytosis of secretory IgA molecules to mount a gut immune response.

Cloning of an aminoglycoside-resistance-encoding gene, kamC, from Saccharopolyspora hirsuta: comparison with kamB from Streptomyces tenebrarius.

An aminoglycoside-resistance-encoding gene (kamC) has been isolated from the sporaricin producer, Saccharopolyspora (Sac.) hirsuta, and expressed both in Streptomyces lividans and Escherichia coli. The pattern of resistance conferred by this gene was identical to that given by another gene (kamB) previously isolated from Streptomyces tenebrarius. In accordance with the known action of the kamB product, the Sac, hirsuta determinant also encodes a methyltransferase that modifies 16S rRNA, thereby rendering ribosomes refractory to certain aminoglycosides. The nucleotide sequences of both genes have been determined and comparison of the deduced amino acid sequences reveals a high degree of similarity.

Cloning, Expression in Escherichia coli and Nucleotide Sequence of a Tetracycline-resistance Gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.

IgG1 Allotypes Influence the Pharmacokinetics of Therapeutic Monoclonal Antibodies through FcRn Binding

Because IgG1 allotypes might have different half-lives, their influence on infliximab (G1m17,1 allotype) pharmacokinetics was investigated in a group of spondyloarthritis patients. Infliximab was found to have a shorter half-life in patients homozygous for the G1m17,1 allotypes than in those carrying the G1m3 with no G1m1 (G1m3,-1) allotype. Because the neonatal FcR (FcRn) is involved in the pharmacokinetics of mAbs, the interaction of different IgG1 allotypes with FcRn was examined using cellular assays and surface plasmon resonance. G1m17,1 mAbs, such as infliximab and rituximab, were shown to bind more efficiently to FcRn and to be transcytosed better than the G1m3,-1 mAb cetuximab, which explains why infliximab is a better competitor for endogenous IgG1 in G1m3,-1 allotype–bearing patients. A set of four allotype variants of adalimumab (G1m17,1; G1m17,-1; G1m3,1; and G1m3,-1) was also tested for its binding to FcRn, revealing that the G1m3,1 variant, not present in commercial mAbs, binds more efficiently to FcRn and is transcytosed better than the other three variants, all of which are found in therapeutic mAbs.

Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells

Background While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1. Design and Methods In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells. Results We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils. Conclusions We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

SummaryXylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.

Rational design of adjuvants targeting the C-type lectin Mincle

Significance For many years, the development of adjuvants—compounds that boost the immunogenicity of vaccines—has been an empirical process. Adjuvants inducing a strong humoral immunity are available, but adjuvants directing the development of robust cellular immune responses are still needed. Recently, the C-type lectin receptor Mincle was found to elicit such responses on the recognition of microbial glycolipids, thereby providing a basis for the rational design of new adjuvants. Here we used a multidisciplinary approach, combining chemical synthesis, biology, and molecular modeling to decipher the molecular bases of ligand recognition by the receptor. This led us to synthesize new compounds inducing stronger immune responses than the currently available Mincle ligands that represent new powerful adjuvant molecules. The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6′-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O-6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties.

Production and characterization of chimeric anti-HLA monoclonal antibodies targeting public epitopes as tools for standardizations of the anti-HLA antibody detection

Two chimeric monoclonal antibodies (MoAbs) were designed with variable parts of the mouse antibodies W6/32 and F3.3, and the human constant parts C-gamma1 and C-kappa. These chimeric MoAbs are specific for HLA-class I and HLA-class II public epitopes, respectively. The anti-class I MoAb recognizes all HLA-class I tested so far, and the anti-class II MoAb recognize all HLA-DR, DP, and only DQ antigens of the DQ2 subgroup. These Moabs were used as positive controls in routine tests for the detection of IgG anti-HLA antibodies in the sera of patients (Luminex, flow cytometry, and complement-dependent lymphocytotoxicity assay: CD-LCT). In tests with the LABScreen MIX assay, serial dilutions of the two MoAbs have allowed to determine the thresholds of detection by the Boltzmann sigmoidal regression. The thresholds of detection in mean of fluorescence intensity (MFI) were 926 and 866 for the anti-class I MoAb and the anti-class II MoAb, respectively. The thresholds defined as mean+3SD of MFI values obtained with 30 negative control sera were 411 and 251 for the anti-class I beads and the anti-class II beads, respectively. For the daily validation of the anti-HLA IgG antibodies screening by LABScreen MIX we decided to use the positive control MoAbs at three concentrations (5, 50, and 500ng/ml) and we measured the repeatability (<10%) and reproducibility (<16%) of the method. Used as positive controls in tests with the LABScreen Single Antigen kit, the two MoAbs allowed to estimate daily the quality of beads coated with HLA antigens. The two anti HLA MoAbs were also used as positive control in the cross-match assay by flow cytometry or CD-LCT. In total these two chimeric anti-HLA-MoAbs, which have no equivalent so far, are valuable positive controls for the daily validations of most techniques used for the detection of anti-HLA antibodies according to the good practice guidelines for laboratories.

Long-lasting In vivo Gene Silencing by Electrotransfer of shRNA Expressing Plasmid

RNA interference appears as a promising tool for therapeutic gene silencing. A key limit is the delivery of the siRNA. A safe approach is to use a physical method such as in vivo electropulsation with contact electrodes. Getting a long lived silencing can be better approached by using the in situ expression of shRNA. This is presently obtained by using co-electrotransfer of specific plasmids coding for expression and silencing of a fluorescent protein. Using a non invasive fluorescence imaging assay, electrodelivery in mouse muscles is observed to induce complete silencing over more than two months in a specific way. The proper choices of the plasmids (sequence and relative amounts) and of the electric pulsing conditions appear as key parameters in the successful silencing.