Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis

Abstract Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia -specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.     

Immunobiological studies with mycobacterial ribonucleic acid-protein complex: Part I--Immune responses and protective role against experimental tuberculosis.

Ribonucleic acid (RNA) isolated from M. tuberculosis H37Ra was found to be native in nature as determined by hyperchromicity studies using ribonuclease. Mycobacterial RNA-protein (Myc. RNA-P) when injected as RNA-P-FIA complexes induced weak humoral immune responses and strong cell-mediated immune (CMI) responses which were directed against Myc. RNA. Protection comparable to BCG was induced in mice immunized with RNA-FIA complexes against LD50 dose of M. tuberculosis as monitored by increased survival rates, decreased lung density, root specific lung weight (RSLW) and by decreased viable counts of M. tuberculosis in lung, liver and spleen of immunized mice. Enzymatic degradation studies revealed Myc. RNA component to specifically mediate protection while the protein component was found ineffective.     

Targeted Gene Correction: Synthesis and Characterization of Double-Hairpin 2′-O-Methyl RNA/DNA Chimera Oligonucleotides

Abstract

A series of 2′-O-methyl RNA/DNA chimera oligonucleotides were synthesized with a double-hairpin structural motif. Liposome formulated delivery of the chimeras effected targeted, high conversion of mutant alleles in mammalian cell culture. The chimera oligonucleotides were prepared with DNA and 2′-OMe RNA phosphoramidite nucleoside monomers on the ABI 394 synthesizer.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Mammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α-, β- or the γ-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk–G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α-, β- and γ-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α- and γ-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.     

Design,synthesis and structure–activity relationships of azole acids as novel,potent dual PPAR α/γ agonists

The design, synthesis and structure–activity relationships of a novel series of N-phenyl-substituted pyrrole, 1,2-pyrazole and 1,2,3-triazole acid analogs as PPAR ligands are outlined. The triazole acid analogs 3f and 4f were identified as potent dual PPARα/γ agonists both in binding and functional assays in vitro. The 3-oxybenzyl triazole acetic acid analog 3f showed excellent glucose and triglyceride lowering in diabetic db/db mice.     

Comparative Genome-Scale Metabolic Reconstruction and Flux Balance Analysis of Multiple Staphylococcus aureus Genomes Identify Novel Antimicrobial Drug Targets

Mortality due to multidrug-resistant Staphylococcus aureus infection is predicted to surpass that of human immunodeficiency virus/AIDS in the United States. Despite the various treatment options for S. aureus infections, it remains a major hospital- and community-acquired opportunistic pathogen. With the emergence of multidrug-resistant S. aureus strains, there is an urgent need for the discovery of new antimicrobial drug targets in the organism. To this end, we reconstructed the metabolic networks of multidrug-resistant S. aureus strains using genome annotation, functional-pathway analysis, and comparative genomic approaches, followed by flux balance analysis-based in silico single and double gene deletion experiments. We identified 70 single enzymes and 54 pairs of enzymes whose corresponding metabolic reactions are predicted to be unconditionally essential for growth. Of these, 44 single enzymes and 10 enzyme pairs proved to be common to all 13 S. aureus strains, including many that had not been previously identified as being essential for growth by gene deletion experiments in S. aureus. We thus conclude that metabolic reconstruction and in silico analyses of multiple strains of the same bacterial species provide a novel approach for potential antibiotic target identification.Staphylococcus aureus is a major hospital/community-acquired opportunistic pathogen. It causes bacteremia, pneumonia, endocarditis, meningitis, and toxic-shock syndrome in adult humans; skin lesions, impetigo, and abscesses in children; and mastitis in cattle (7, 22, 27). In general, S. aureus infections are treated with β-lactam antibiotics, sulfa drugs, tetracycline, and clindamycin. However, drug-resistant strains, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have emerged from both hospital and community infections in recent years. To date, only one new drug candidate, platensimycin, has been found to be effective against some strains of MRSA and VRSA (30). A recent meta-analysis suggested that mortality due to multidrug-resistant S. aureus in the United States may exceed that from human immunodeficiency virus infections and AIDS (19). This has resulted in a renewed interest in identifying new targets and molecules effective against multidrug-resistant strains of bacteria, and S. aureus in particular.Based on whole-genome sequence comparisons, S. aureus strains can be divided into three divergent groups arising from a common lineage (11). Significant sequence variations between animal and human S. aureus strains have also been identified (15). Though many virulence and drug resistance markers have been studied, the cause of the continuous emergence of multidrug-resistant strains remains elusive, as the resistance phenotype is not attributable to a few studied genes. Combining the data from multilocus sequence typing, microarray analysis, sequence relationships, homologous recombination, and phages of S. aureus, two major groups of clonal strains have been identified (11). A similar conclusion was reached when the S. aureus Newman genome sequence was compared to those of 11 other S. aureus strains (3). These studies not only confirm the clonality of the genome, but also reveal that nearly 20% of the sequence variations are due to prophages and pathogenicity islands.In order to further refine a generic antimicrobial drug target identification scheme (2), we performed metabolic reconstructions of multidrug-resistant and sensitive strains of S. aureus. This was feasible, as the genome sequences of 13 S. aureus strains are now available. They include strain N315 (a MRSA strain), Mu50 (a VRSA strain), JH9 (a vancomycin-nonsusceptible MRSA strain), JH1 (a vancomycin-susceptible, hospital-acquired MRSA strain), COL (a hospital-acquired MRSA strain), 252 (a hospital-acquired MRSA strain), USA 300 (a community-acquired MRSA strain), MW2 (a community-acquired MRSA strain), and RF122 (a bovine mastitis strain).Previous efforts in the metabolic reconstruction and subsequent flux balance analysis (FBA) of a single S. aureus strain (N315) provided valuable but limited insight into the metabolic capabilities of the bacterium (4, 14). Using this strain (20), Becker and Palsson predicted 518 metabolic reactions and 571 metabolites based on a limited set of genes (enzymes) (4). Their study also identified the components of minimal growth medium for S. aureus. Of the six required amino acids, only four (l-alanine, l-arginine, l-proline, and l-glycine) were common to both experimental and computational studies. Glucose (carbon source), phosphate, sulfate, nicotinamide, and thiamine were both experimentally utilized and computationally verified. However, other substrates, such as the nucleosides cytidine and uridine, were predicted not to be required in their metabolic model. A second genome-scale reconstruction of the same strain based on the KEGG ligand database was carried out and yielded 774 metabolic reactions catalyzed by 394 unique enzymes (13). Heinemann et al. (14) also validated their reconstruction using published experimental data and further defined a biomass composition for S. aureus.To reconcile the results of these two previous reconstructions and to address the differences in the metabolic capabilities of various S. aureus strains, we employed comprehensive genomic and metabolic reconstruction methodologies using the ERGO bioinformatics suite (24). This approach enabled us to identify the functional pathways, metabolic reactions, and transport reactions of several sequenced strains of S. aureus. The identified metabolic pathways and their individual reactions were systematically compared with those archived in the KEGG ligand database (17). FBA of such reconstructed metabolic networks have allowed in silico single and double gene deletion experiments, e.g., in Escherichia coli (2, 8, 23, 29). The application of these methods has led us to the identification of single enzymes and synthetic enzyme pairs that are unconditionally required for the growth (biomass production) of all S. aureus strains.     

TLR-activated dendritic cells enhance the response of aged naive CD4 T cells via an IL-6-dependent mechanism

The most effective immunological adjuvants contain microbial products, such as TLR agonists, which bind to conserved pathogen recognition receptors. These activate dendritic cells (DCs) to become highly effective APCs. We assessed whether TLR ligand-treated DCs can enhance the otherwise defective response of aged naive CD4 T cells. In vivo administration of CpG, polyinosinic-polycytidylic acid, and Pam(3)CSK(4) in combination with Ag resulted in the increased expression of costimulatory molecules and MHC class II by DCs, increased serum levels of the inflammatory cytokines IL-6 and RANTES, and increased cognate CD4 T cell responses in young and aged mice. We show that, in vitro, preactivation of DCs by TLR ligands makes them more efficient APCs for aged naive CD4 T cells. After T-DC interaction, there are enhanced production of inflammatory cytokines, particularly IL-6, and greater expansion of the aged T cells, resulting from increased proliferation and greater effector survival with increased levels of Bcl-2. TLR preactivation of both bone marrow-derived and ex vivo DCs improved responses. IL-6 produced by the activated DCs during cognate T cell interaction was necessary for enhanced aged CD4 T cell expansion and survival. These studies suggest that some age-associated immune defects may be overcome by targeted activation of APCs by TLR ligands.     

Chloroquine treatment increases detection of 5-fluorouracil-induced apoptosis index in vivo

Although apoptosis can be readily assessed in vitro with a variety of techniques, the detection of apoptosis in the in vivo setting poses a much more difficult proposition. Apoptosis in an organism is followed almost inevitably by rapid clearance of dying cells via phagocytosis, thus limiting the ability to analyze apoptosis in vivo using classical techniques. To address this issue, we developed a method to enhance in vivo apoptosis detection using pretreatment with chloroquine, an inhibitor of macrophage activity, in Swiss albino mice. This technique resulted in a significant increase in the accumulation of apoptotic cells induced by 5-fluorouracil, as detected by propidium iodide staining in solid and ascitic forms of Ehrlich ascitic tumors and in bone marrow cells. We further validated our technique using DNA fragmentation and endonuclease assays. Our results demonstrated that chloroquine pretreatment can significantly enhance accumulation of apoptotic cells in organisms, and we envision combining this method with modern imaging techniques to optimize in vivo detection of apoptosis.