The individual contributions of blaB, blaGOB and blaCME on MICs of ß-lactams in Elizabethkingia anophelis.

BACKGROUND: The blaB, blaGOB and blaCME genes are thought to confer ß-lactam resistance to Elizabethkingia anophelis, based on experiments conducted primarily on Escherichia coli. OBJECTIVES: To determine the individual contributions of ß-lactamase genes to increased MICs in E. anophelis and to assess their impact on the in vivo efficacy of carbapenem therapy. METHODS: Scarless gene deletion of one or more ß-lactamase gene(s) was performed in three clinical E. anophelis isolates. MICs were determined by broth microdilution. Hydrolytic activity and expressions of ß-lactamase genes were measured by an enzymatic assay and quantitative RT-PCR, respectively. In vivo efficacy was determined using Galleria mellonella and murine thigh infection models. RESULTS: The presence of blaB resulted in >16-fold increases, while blaGOB caused 4-16-fold increases of carbapenem MICs. Hydrolysis of carbapenems was highest in lysates of blaB-positive strains, possibly due to the constitutionally higher expression of blaB. Imipenem was ineffective against blaB-positive isolates in vivo in terms of improvement of the survival of wax moth larvae and reduction of murine bacterial load. The deletion of blaB restored the efficacy of imipenem. The blaB gene was also responsible for a >4-fold increase of ampicillin/sulbactam and piperacillin/tazobactam MICs. The presence of blaCME, but not blaB or blaGOB, increased the MICs of ceftazidime and cefepime by 8-16- and 4-8-fold, respectively. CONCLUSIONS: The constitutionally and highly expressed blaB gene in E. anophelis was responsible for increased MICs of carbapenems and led to their poor in vivo efficacy. blaCME increased the MICs of ceftazidime and cefepime.

Aqueous Ocimum gratissimum extract induces cell apoptosis in human hepatocellular carcinoma cells.

Treatment of advanced hepatocellular carcinoma (HCC) has exhibited a poor overall survival rate of only six to ten months, and the urgency of the development of more effective novel agents is ever present. In this line of research, we aimed to investigate the effects and inhibitive mechanisms of aqueous Ocimum gratissimum leaf extract (OGE), the extract of Ocimum gratissimum, which is commonly used as a therapeutic herb for its numerous pharmacological properties, on malignant HCC cells. Our results showed that OGE decreased the cell viability of HCC SK-Hep1 and HA22T cells in a dose-dependent manner (from 400 to 800 µg/mL), while there is little effect on Chang liver cells. Moreover, cell-cycle analysis shows increased Sub-G1 cell count in SK-Hep1 and HA22T cells which is not observed in Chang liver cells. These findings raise suspicion that the OGE-induced cell death may be mediated through proteins that regulate cell cycle and apoptosis in SK-Hep1 and HA22T cells, and further experimentation revealed that OGE treatment resulted in a dose-dependent decrease in caspase 3 and PARP expressions and in CDK4and p-ERK1/2expressions. Moreover, animal tests also exhibited decreased HCC tumor growth by OGE treatment. We therefore suggest that the inhibition of cell viability and tumor growth induced by OGE may be correlated to the alteration of apoptosis-related proteins.

Integration of a Thermoelectric Heating Unit with Ionic Wind-Induced Droplet Centrifugation Chip to Develop Miniaturized Concentration Device for Rapid Determination of Salmonella on Food Samples Using Antibody-Functionalized SERS Tags.

When a centrifugation-enriched sample of 100 µL containing the surface-enhanced Raman scattering (SERS) tag-bound bacteria (Salmonella in this study) is siphoned onto a glass slide next to an embedded thermoelectric heating chip, such a sessile droplet is quickly evaporated. As the size of the sample droplet is significantly reduced during the heating process, ionic wind streams from a corona discharge needle, stationed above the sample, sweep across the liquid surface to produce centrifugal vortex flow. Tag-bound Salmonella in the sample are then dragged and trapped at the center of droplet bottom. Finally, when the sample is dried, unlike the "coffee ring" effect, the SERS tag-bound Salmonella is concentrated in one small spot to allow sensitive detection of a Raman signal. Compared with our previous electrohydrodynamic concentration device containing only a corona discharge needle, this thermoelectric evaporation-assisted device is more time-effective, with the time of concentrating and drying about 100 µL sample reduced from 2 h to 30 min. Hence, sample throughput can be accelerated with this device for practical use. It is also more sensitive, with SERS detection of a few cells of Salmonella in neat samples achievable. We also evaluated the feasibility of using this device to detect Salmonella in food samples without performing the culturing procedures. Having spiked a few Salmonella cells into ice cubes and lettuce leaves, we use filtration and ultracentrifugation steps to obtain enriched tag-bound Salmonella samples of 200 µL. After loading an aliquot of 100 µL of sample onto this concentration device, the SERS tag signals from samples of 100 g ice cubes containing two Salmonella cells and 20 g lettuce leaf containing 5 Salmonella cells can be successfully detected.

Development of a novel engineered antibody targeting Neisseria species.

OBJECTIVES: A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv). RESULTS: Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine. CONCLUSIONS: The recombinant scFv could detect Neisseria strains at 106 CFU/ml.

Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics.

Protective antibodies play an essential role in immunity to infection by neutralizing microbes or their toxins and recruiting microbicidal effector functions. Identification of the protective B-cell epitopes, those parts of microbial antigens that contact the variable regions of the protective antibodies, can lead to development of antibody therapeutics, guide vaccine design, enable assessment of protective antibody responses in infected or vaccinated individuals, and uncover or localize pathogenic microbial functions that could be targeted by novel antimicrobials. Monoclonal antibodies are required to link in vivo or in vitro protective effects to specific epitopes and may be obtained from experimental animals or from humans, and their binding can be localized to specific regions of antigens by immunochemical assays. The epitopes are then identified with mapping methods such as X-ray crystallography of antigen-antibody complexes, antibody inhibition of hydrogen-deuterium exchange in the antigen, antibody-induced alteration of the nuclear magnetic resonance spectrum of the antigen, and experimentally validated computational docking of antigen-antibody complexes. The diversity in shape, size and structure of protective B-cell epitopes, and the increasing importance of protective B-cell epitope discovery to development of vaccines and antibody therapeutics are illustrated through examples from different microbe categories, with emphasis on epitopes targeted by broadly neutralizing antibodies to pathogens of high antigenic variation. Examples include the V-shaped Ab52 glycan epitope in the O-antigen of Francisella tularensis, the concave CR6261 peptidic epitope in the haemagglutinin stem of influenza virus H1N1, and the convex/concave PG16 glycopeptidic epitope in the gp120 V1/V2 loop of HIV type 1.

On-line SERS detection of single bacterium using novel SERS nanoprobes and a microfluidic dielectrophoresis device.

The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfluidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica. The SERS nanoprobes are prepared by immobilization of specific antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defined plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identification of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB-DEP-Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL(-1) . In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.

A Label-Free Droplet Sorting Platform Integrating Dielectrophoretic Separation for Estimating Bacterial Antimicrobial Resistance.

Antimicrobial resistance (AMR) has become a crucial global health issue. Antibiotic-resistant bacteria can survive after antibiotic treatments, lowering drug efficacy and increasing lethal risks. A microfluidic water-in-oil emulsion droplet system can entrap microorganisms and antibiotics within the tiny bioreactor, separate from the surroundings, enabling independent assays that can be performed in a high-throughput manner. This study presents the development of a label-free dielectrophoresis (DEP)-based microfluidic platform to sort droplets that co-encapsulate Escherichia coli (E. coli) and ampicillin (Amp) and droplets that co-encapsulate Amp-resistant (AmpR) E. coli with Amp only based on the conductivity-dependent DEP force (FDEP) without the assistance of optical analyses. The 9.4% low conductivity (LC) Luria-Bertani (LB) broth diluted with 170 mM mannitol can maintain E. coli and AmpR E. coli growth for 3 h and allow Amp to kill almost all E. coli, which can significantly increase the LCLB conductivity by about 100 µS/cm. Therefore, the AmpR E. coli/9.4%LCLB/Amp where no cells are killed and the E. coli/9.4%LCLB/Amp-containing droplets where most of the cells are killed can be sorted based on this conductivity difference at an applied electric field of 2 MHz and 100 Vpp that generates positive FDEP. Moreover, the sorting ratio significantly decreased to about 50% when the population of AmpR E. coli was equal to or higher than 50% in droplets. The conductivity-dependent DEP-based sorting platform exhibits promising potential to probe the ratio of AmpR E. coli in an unknown bacterial sample by using the sorting ratio as an index.

A Critical Review on Detection of Foodborne Pathogens Using Electrochemical Biosensors.

An outbreak of foodborne pathogens would cause severe consequences. Detecting and diagnosing foodborne diseases is crucial for food safety, and it is increasingly important to develop fast, sensitive, and cost-effective methods for detecting foodborne pathogens. In contrast to traditional methods, such as medium-based culture, nucleic acid amplification test, and enzyme-linked immunosorbent assay, electrochemical biosensors possess the advantages of simplicity, rapidity, high sensitivity, miniaturization, and low cost, making them ideal for developing pathogen-sensing devices. The biorecognition layer, consisting of recognition elements, such as aptamers, antibodies and bacteriophages, and other biomolecules or polymers, is the most critical component to determine the selectivity, specificity, reproducibility, and lifetime of a biosensor when detecting pathogens in a biosample. Furthermore, nanomaterials have been frequently used to improve electrochemical biosensors for sensitively detecting foodborne pathogens due to their high conductivity, surface-to-volume ratio, and electrocatalytic activity. In this review, we survey the characteristics of biorecognition elements and nanomaterials in constructing electrochemical biosensors applicable for detecting foodborne pathogens during the past five years. As well as the challenges and opportunities of electrochemical biosensors in the application of foodborne pathogen detection are discussed.

The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide.

We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions.

Genome, integration, and transduction of a novel temperate phage of Helicobacter pylori.

Helicobacter pylori is a common human pathogen that has been identified to be carcinogenic. This study isolated the temperate bacteriophage 1961P from the lysate of a clinical strain of H. pylori isolated in Taiwan. The bacteriophage has an icosahedral head and a short tail, typical of the Podoviridae family. Its double-stranded DNA genome is 26,836 bp long and has 33 open reading frames. Only 9 of the predicted proteins have homologs of known functions, while the remaining 24 are only similar to unknown proteins encoded by Helicobacter prophages and remnants. Analysis of sequences proximal to the phage-host junctions suggests that 1961P may integrate into the host chromosome via a mechanism similar to that of bacteriophage lambda. In addition, 1961P is capable of generalized transduction. To the best of our knowledge, this is the first report of the isolation, characterization, genome analysis, integration, and transduction of a Helicobacter pylori phage.

Infrequent cross-transmission of Shigella flexneri 2a strains among villages of a mountainous township in Taiwan with endemic shigellosis.

BACKGROUND: Shigellosis is rare in Taiwan, with an average annual incidence rate of 1.68 cases per 100,000 persons in 2000-2007. However, the incidence rate for a mountainous township in eastern Taiwan, Zhuoxi, is 60.2 times the average rate for the entire country. Traveling between Zhuoxi's 6 villages (V1-V6) is inconvenient. Disease transmission among the villages/tribes with endemic shigellosis was investigated in this study. METHODS: Demographic data were collected in 2000-2010 for epidemiological investigation. Thirty-eight Shigella flexneri 2a isolates were subjected to pulsed-field gel electrophoresis (PFGE) genotyping and antimicrobial susceptibility testing (AST). RESULTS: Fifty-five shigellosis cases were identified in 2000-2007, of which 38 were caused by S. flexneri 2a from 2000-2007, 16 cases were caused by S. sonnei from 2000-2003, and 1 case was caused by S. flexneri 3b in 2006. S. flexneri 2a caused infections in 4 of the 6 villages of Zhuoxi Township, showing the highest prevalence in villages V2 and V5. PFGE genotyping categorized the 38 S. flexneri 2a isolates into 2 distinct clusters (clones), 1 and 2. AST results indicated that most isolates in cluster 1 were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and trimethoprim-sulfamethoxazole (ACSSuX); all isolates in cluster 2 were resistant to ACSSuX and tetracycline. Genotypes were primarily unique to different villages or tribes. Tribe V2-1 showed the highest endemic rates. Eighteen isolates recovered from V2-1 tribe members fell into 6 genotypes, where 5 were the same clone (cluster 1). An outbreak (OB2) in 2004 in village V2 was caused by different clonal strains; cases in tribe V2-1 were caused by 2 strains of clone 1, and those in tribe V2-2 were infected by a strain of clone 2. CONCLUSIONS: From 2000-2007, 2 S. flexneri 2a clones circulated among 4 villages/tribes in the eastern mountainous township of Zhuoxi. Genotyping data showed restricted disease transmission between the villages and tribes, which may be associated with difficulties in traveling between villages and limited contact between different ethnic aborigines. Transmission of shigellosis in this township likely occurred via person-to-person contact. The endemic disease was controlled by successful public health intervention.

Luteolin inhibits migration of human glioblastoma U-87 MG and T98G cells through downregulation of Cdc42 expression and PI3K/AKT activity.

Luteolin (3',4',5,7-tetrahydroxyflavone) is a common flavonoid in many types of plants and has several beneficial biological effects, including anti-inflammation, anti-oxidant, and anti-cancer properties. However, the detail mechanisms of luteolin in suppressing tumor invasion and metastasis are poorly understood. Here, we investigated the effects of luteolin on suppressing glioblastoma tumor cell invasion and migration activity. Under the non-cytotoxic doses (15 and 30 µM), luteolin exhibited an inhibitory effect on migration and invasion in U-87 MG and T98G glioblastoma cells. Additionally, filopodia assembly in U-87 MG cells was markedly suppressed after luteolin treatment. The treatment of luteolin also showed a decrease of Cdc42 (cell division cycle 42) protein levels and reduced PI3K/AKT activation, whereas there was no association between this decrease and phosphorylated ERK or altered transcription levels of Cdc42. Over expression of constitutive Cdc42 (Q61L) using transient transfection in U-87 MG cells induced a partial cell migration, but did not affected the degradation of the protein levels of Cdc42 after luteolin treatment. Moreover, inhibition of the proteaosome pathway by MG132 caused a significant recovery in the migration ability of U-87 MG cells and augmented the Cdc42 protein levels after luteolin treatment, suggesting that pharmacological inhibition of migration via luteolin treatment is likely to preferentially facilitate the protein degradation of Cdc42. Taken together, the study demonstrated that flavonoids of luteolin prevent the migration of glioblastoma cells by affecting PI3K/AKT activation, modulating the protein expression of Cdc42 and facilitating their degradation via the proteaosome pathway.

Immunohistochemical Stain-Aided Annotation Accelerates Machine Learning and Deep Learning Model Development in the Pathologic Diagnosis of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial cancer originating in the nasopharynx epithelium. Nevertheless, annotating pathology slides remains a bottleneck in the development of AI-driven pathology models and applications. In the present study, we aim to demonstrate the feasibility of using immunohistochemistry (IHC) for annotation by non-pathologists and to develop an efficient model for distinguishing NPC without the time-consuming involvement of pathologists. For this study, we gathered NPC slides from 251 different patients, comprising hematoxylin and eosin (H&E) slides, pan-cytokeratin (Pan-CK) IHC slides, and Epstein-Barr virus-encoded small RNA (EBER) slides. The annotation of NPC regions in the H&E slides was carried out by a non-pathologist trainee who had access to corresponding Pan-CK IHC slides, both with and without EBER slides. The training process utilized ResNeXt, a deep neural network featuring a residual and inception architecture. In the validation set, NPC exhibited an AUC of 0.896, with a sensitivity of 0.919 and a specificity of 0.878. This study represents a significant breakthrough: the successful application of deep convolutional neural networks to identify NPC without the need for expert pathologist annotations. Our results underscore the potential of laboratory techniques to substantially reduce the workload of pathologists.

Carboxy-Terminal Processing Protease Controls Production of Outer Membrane Vesicles and Biofilm in Acinetobacter baumannii.

Carboxy-terminal processing protease (Ctp) is a serine protease that controls multiple cellular processes through posttranslational modification of proteins. Acinetobacter baumannii ATCC 17978 ctp mutant, namely MR14, is known to cause cell wall defects and autolysis. The objective of this study was to investigate the role of ctp mutation-driven autolysis in regulating biofilms in A. baumannii and to evaluate the vesiculation caused by cell wall defects. We found that in A. baumannii, Ctp is localized in the cytoplasmic membrane, and loss of Ctp function enhances the biofilm-forming ability of A. baumannii. Quantification of the matrix components revealed that extracellular DNA (eDNA) and proteins were the chief constituents of MR14 biofilm, and the transmission electron microscopy further indicated the presence of numerous dead cells compared with ATCC 17978. The large number of MR14 dead cells is potentially the result of compromised outer membrane integrity, as demonstrated by its high sensitivity to sodium dodecyl sulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). MR14 also exhibited the hypervesiculation phenotype, producing outer-membrane vesicles (OMVs) of large mean size. The MR14 OMVs were more cytotoxic toward A549 cells than ATCC 17978 OMVs. Our overall results indicate that A. baumanniictp negatively controls pathogenic traits through autolysis and OMV biogenesis.

Immunoproteomic identification of the hypothetical protein NMB1468 as a novel lipoprotein ubiquitous in Neisseria meningitidis with vaccine potential.

Many potential vaccine candidates for serogroup B Neisseria meningitidis (NMB) have been identified by reverse vaccinology, a genome-based approach. However, some candidates may be unseen owing to uncertain annotation or their peculiar properties. In this study, we describe the preparation and identification of a novel lipoprotein expressed in all meningococcal strains tested. mAb were first prepared from mice immunized with a meningococcal B strain isolated in Taiwan. Total proteins from the immunizing strain were separated by 2-DE in duplicate. Clone 4-7-3, which crossreacted to 174 tested meningococcal isolates, was used as the primary antibody for Western blotting. The immunoreactive spot was identified by LC-mass spectrometric analysis of the corresponding spot from the silver-stained gel and confirmed by molecular biology approach to be a novel lipoprotein encoded by the hypothetical NMB1468 gene. The potential use of this protein, designated Ag473/NMB1468, as a vaccine component was evaluated using the recombinant protein produced in Escherichia coli. Immunized mice were found to be protected from developing meningococcal disease after intraperitoneal inoculation with a lethal dose of meningococcal strain Nm22209, suggesting that Ag473/NMB1468 may be a promising vaccine candidate. This study also demonstrates the usefulness of the immunoproteomic approach in identification of novel vaccine candidates.

Rhein induces apoptosis through induction of endoplasmic reticulum stress and Ca2+-dependent mitochondrial death pathway in human nasopharyngeal carcinoma cells.

Apoptosis is a physiological mechanism for eliminating malignant cells, including cancer cells, without eliciting damage to normal cells or surrounding tissues. Here, we report that rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), a major constituent in the rhizome of rhubarb, induced apoptosis of human nasopharyngeal carcinoma (NPC) cells. Rhein induced apoptosis in NPC cells as demonstrated by increased nuclear condensation and DNA fragmentation. Moreover, for the first time in NPC cells it was demonstrated that the pathway involved in rhein-induced apoptosis is caspase-dependent, presumably through the endoplasmic reticulum (ER) stress pathway, as shown by an increase in the levels of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), activating transcription factor 6 (A TF6) and CCAA TIenhancer-binding protein homologous protein (CHOP) as well as the activation of caspase-3, -8, -9 and -12. This increased susceptibility to ER stress-induced apoptosis may be due to an increased accumulation of reactive oxygen species (ROS). Rapid accumulation of calcium (Ca2+) and a decrease in the mitochondrial membrane potential (MMP) were also observed. Cytochrome c and apoptosis-inducing factor (AIF) were released upon treatment with rhein. Taken together, these results suggest that ER stress and Ca2+-dependent mitochondrial death pathway may be involved in rhein-induced apoptosis in NPC cells.

Identification of the antigenic determinants of the American cockroach allergen Per a 1 by error-prone PCR.

The group I allergen of cockroach is found in both American and German cockroaches, designated as Per a 1 and Bla g 1, respectively. Members of these allergens so far identified are composed of tandem repeats that may cause the high allergenicity of Per a 1 allergen. In this study, we used monoclonal antibodies HW-8 and HW-19, which can inhibit the binding of patient IgE to Per a 1 allergen, to define the structure of the antigenic determinants in Per a 1.0103 (designated C3), an isoallergen of Per a 1 allergen. Two recognition sites are present, one in the N-terminus (aa 1-208) and the other in the C-terminus (aa 208-395). The N-terminal epitope is not accessible to antibody molecules on the pET-expressed C3 protein. The C-terminal epitope was further localized to the aa 267-354 region (C3E) by colony immunoscreening of the cDNA epitope library. By negative screening of the mutated C3E expression library generated by error-prone PCR (ER-PCR), an approach which has rarely been applied in epitope mapping, the functional epitope was identified to lie in aa 318-337 with aa 323-331 being the core motif. The minimal region of the functional epitope was further delineated, by sequence alignment, to be D-x-[I, L]-A-[I, L]-L-P-V-D-E-[L, I]-x-A-[L, I], where x represents any amino acids. This motif is found in all Per a 1 allergens and may serve as a basis for designing a peptide vaccine for allergen-specific immunotherapy. To our knowledge, this is the first report for (1) detailed mapping of the cockroach allergens and (2) use of error-prone PCR random mutagenesis and negative selection in molecular allergology.

Polyclonal Fab phage display libraries with a high percentage of diverse clones to Cryptosporidium parvum glycoproteins.

The protozoan parasite Cryptosporidium parvum is regarded as a major public health problem world-wide, especially for immunocompromised individuals. Although no effective therapy is presently available, specific immune responses prevent or terminate cryptosporidiosis and passively administered antibodies have been found to reduce the severity of infection. Therefore, as an immunotherapeutic approach against cryptosporidiosis, we set out to develop C. parvum-specific polyclonal antibody libraries, standardised, perpetual mixtures of polyclonal antibodies, for which the genes are available. A combinatorial Fab phage display library was generated from the antibody variable region gene repertoire of mice immunised with C. parvum surface and apical complex glycoproteins which are believed to be involved in mediating C. parvum attachment and invasion. The variable region genes used to construct this starting library were shown to be diverse by nucleotide sequencing. The library was subjected to one round of antigen selection on C. parvum glycoproteins or a C. parvum oocyst/sporozoite preparation. The two selected libraries showed specific reactivity to the glycoproteins as well as to the oocyst/sporozoite preparation, with 50-73% antigen-reactive members. Fingerprint analysis of individual clones from the two antigen-selected libraries showed high diversity, confirming the polyclonality of the selected libraries. Furthermore, immunoblot analysis on the oocyst/sporozoite and glycoprotein preparations with selected library phage showed reactivity to multiple bands, indicating diversity at the antigen level. These C. parvum-specific polyclonal Fab phage display libraries will be converted to libraries of polyclonal full-length antibodies by mass transfer of the selected heavy and light chain variable region gene pairs to a mammalian expression vector. Such polyclonal antibody libraries would be expected to mediate effector functions and provide optimal passive immunity against cryptosporidiosis.

Functional and structural characterization of Francisella tularensis O-antigen antibodies at the low end of antigen reactivity.

The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them. In a mouse model of respiratory tularemia with the highly virulent Ft type A strain SchuS4, the three new MAbs reduced blood bacterial burden with potencies that mirror their antigen-binding strength; the best binder of the new MAbs, N203, prolonged survival in a dose-dependent manner, but was at least 10-fold less potent than the best previously characterized IgG2a MAb, Ab52. X-ray crystallographic studies of N203 Fab showed a flexible binding site in the form of a partitioned groove, which cannot provide as many contacts to OAg as does the Ab52 binding site. These results reveal structural features of antibodies at the low end of reactivity with multi-repeat microbial carbohydrates and demonstrate that such antibodies still have substantial protective effects against infection.

B-cell epitopes in GroEL of Francisella tularensis.

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other's binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange-mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL.