scholarly journals Expression of Cytolethal Distending Toxin and Hemolysin Is Not Required for Pustule Formation by Haemophilus ducreyi in Human Volunteers

2001 ◽  
Vol 69 (3) ◽  
pp. 1938-1942 ◽  
Author(s):  
Royden S. Young ◽  
Kate R. Fortney ◽  
Valentina Gelfanova ◽  
Carrie L. Phillips ◽  
Barry P. Katz ◽  
...  
Keyword(s):  
Double Mutant ◽  
Human Subjects ◽  
Haemophilus Ducreyi ◽  
Cytolethal Distending Toxin ◽  
Deficient Mutant ◽  
Human Volunteers

ABSTRACT Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.

scholarly journals Expression of the LspA1 and LspA2 Proteins by Haemophilus ducreyi Is Required for Virulence in Human Volunteers

2004 ◽  
Vol 72 (8) ◽  
pp. 4528-4533 ◽  
Author(s):  
Diane M. Janowicz ◽  
Kate R. Fortney ◽  
Barry P. Katz ◽  
Jo L. Latimer ◽  
Kaiping Deng ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Double Mutant ◽  
Human Subjects ◽  
Rabbit Model ◽  
Haemophilus Ducreyi ◽  
Wild Type ◽  
Human Volunteers ◽  
Mean Size ◽  
Wild Type Parent

ABSTRACT Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HPΩ12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm2) than at parent sites (mean size, 39.1 mm2) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

scholarly journals Expression of Sialylated or Paragloboside-Like Lipooligosaccharides Are Not Required for Pustule Formation byHaemophilus ducreyi in Human Volunteers

1999 ◽  
Vol 67 (12) ◽  
pp. 6335-6340 ◽  
Author(s):  
Royden S. Young ◽  
Kate Fortney ◽  
Jennifer C. Haley ◽  
Antoinette F. Hood ◽  
Anthony A. Campagnari ◽  
...  
Keyword(s):  
Dose Response ◽  
Preliminary Data ◽  
Human Subjects ◽  
Etiologic Agent ◽  
Haemophilus Ducreyi ◽  
Recovery Rates ◽  
Isogenic Mutant ◽  
Human Volunteers ◽  
A Minor ◽  
Response Trial

ABSTRACT The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens. To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed inlosB, which encodesd-glycero-d-manno-heptosyltransferase. 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid. Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial. For estimated delivered doses (EDDs) of ≥25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2. Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require dd-heptose to form the terminal N-acetyllactosamine. If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform. To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects. For EDDs of ≥25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar. These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H. ducreyi in humans.

scholarly journals DsrA-Deficient Mutant of Haemophilus ducreyi Is Impaired in Its Ability To Infect Human Volunteers

2001 ◽  
Vol 69 (3) ◽  
pp. 1488-1491 ◽  
Author(s):  
Cliffton T. H. Bong ◽  
Robert E. Throm ◽  
Kate R. Fortney ◽  
Barry P. Katz ◽  
Antoinette F. Hood ◽  
...  
Keyword(s):  
Human Subjects ◽  
Formation Rate ◽  
The Other ◽  
Haemophilus Ducreyi ◽  
Surface Areas ◽  
Resistant Phenotype ◽  
Human Volunteers ◽  
Normal Human ◽  
In Trans

ABSTRACT Haemophilus ducreyi produces an outer membrane protein called DsrA, which is required for serum resistance. An isogenicdsrA mutant, FX517, was constructed previously in H. ducreyi 35000. Compared to its parent, FX517 cannot survive in normal human serum. When complemented in trans with a plasmid containing dsrA, FX517 is converted to a serum-resistant phenotype (C. Elkins, K. J. Morrow, Jr., and B. Olsen, Infect. Immun. 68:1608–1619, 2000). To test whetherdsrA was transcribed in vivo, we successfully amplified transcripts in five biopsies obtained from four experimentally infected human subjects. To test whether DsrA was required for virulence, six volunteers were experimentally infected with 35000 and FX517 and observed for papule and pustule formation. Each subject was inoculated with two doses (70 to 80 CFU) of live 35000 and 1 dose of heat-killed bacteria on one arm and with three doses (ranging from 35 to 800 CFU) of live FX517 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent. However, mutant papule surface areas were significantly smaller than parent papules. The pustule formation rate was 58% (95% confidence interval [CI] of 28 to 85%) at 12 parent sites, and 0% (95% CI of 0 to 15%) at 18 mutant sites (P = 0.0004). Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of DsrA facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

scholarly journals A (p)ppGpp-Null Mutant of Haemophilus ducreyi Is Partially Attenuated in Humans Due to Multiple Conflicting Phenotypes

2014 ◽  
Vol 82 (8) ◽  
pp. 3492-3502 ◽  
Author(s):  
Concerta Holley ◽  
Dharanesh Gangaiah ◽  
Wei Li ◽  
Kate R. Fortney ◽  
Diane M. Janowicz ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Parent Strain ◽  
Double Mutant ◽  
Bacterial Species ◽  
Stringent Response ◽  
Haemophilus Ducreyi ◽  
Content Type ◽  
E Coli ◽  
Human Volunteers ◽  
Increased Sensitivity

ABSTRACT(p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species.Haemophilus ducreyi, the causative agent of chancroid, has homologs of bothrelAandspoT, which primarily synthesize and hydrolyze (p)ppGpp inEscherichia coli. We constructedrelAandrelA spoTdeletion mutants to assess the contribution of (p)ppGpp toH. ducreyipathogenesis. Both therelAsingle mutant and therelA spoTdouble mutant failed to synthesize (p)ppGpp, suggesting thatrelAis the primary synthetase of (p)ppGpp inH. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented intrans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription oflspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. InE. coli, overexpression of DksA compensates for the loss of (p)ppGpp; theH. ducreyidouble mutant expressed higher transcript levels ofdksAthan the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.

scholarly journals BIMG-13. A NOVEL RADIOPHARMACEUTICAL ([18F]DASA-23) TO MONITOR PYRUVATE KINASE M2 INDUCED GLYCOLYTIC REPROGRAMMING IN GLIOBLASTOMA

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i3-i4
Author(s):  
Corinne Beinat ◽  
Chirag Patel ◽  
Tom Haywood ◽  
Surya Murty ◽  
Lewis Naya ◽  
...  
Keyword(s):  
Cell Culture ◽  
Pyruvate Kinase ◽  
Cancer Metabolism ◽  
Human Subjects ◽  
Normal Brain ◽  
Healthy Human ◽  
Pyruvate Kinase M2 ◽  
Molar Activity ◽  
Pet Tracer ◽  
Human Volunteers

Abstract BACKGROUND Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain, making it an important biomarker of cancer glycolytic re-programming. We describe the bench-to-bedside development, validation, and translation of a novel positron emission tomography (PET) tracer to study PKM2 in GBM. Specifically, we evaluated 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and GBM patients. METHODS [18F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/µmol and radiochemical purity >95%. We performed initial testing of [18F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next we produced [18F]DASA-23 under current Good Manufacturing Practices United States Food and Drug Administration (FDA) oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with gliomas. RESULTS In mouse imaging studies, [18F]DASA-23 clearly delineated the U87 GBM from the surrounding healthy brain tissue and had a tumor-to-brain ratio (TBR) of 3.6 ± 0.5. In human volunteers, [18F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In GBM patients, [18F]DASA-23 successfully outlined tumors visible on contrast-enhanced magnetic resonance imaging (MRI). The uptake of [18F]DASA-23 was markedly elevated in GBMs compared to normal brain, and it was able to identify a metabolic non-responder within 1-week of treatment initiation. CONCLUSION We developed and translated [18F]DASA-23 as a promising new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These encouraging results warrant further clinical evaluation of [18F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.

scholarly journals Prevalence of cdtABC genes encoding cytolethal distending toxin among Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains

2001 ◽  
Vol 50 (10) ◽  
pp. 860-864 ◽  
Author(s):  
H.J. AHMED ◽  
L.A. SVENSSON ◽  
L.D. COPE ◽  
J.L. LATIMER ◽  
E.J. HANSEN ◽  
...  
Keyword(s):  
Actinobacillus Actinomycetemcomitans ◽  
Haemophilus Ducreyi ◽  
Cytolethal Distending Toxin ◽  
Genes Encoding

scholarly journals A schlieren optical study of the human cough with and without wearing masks for aerosol infection control

2009 ◽  
Vol 6 (suppl_6) ◽  
Author(s):  
Julian W. Tang ◽  
Thomas J. Liebner ◽  
Brent A. Craven ◽  
Gary S. Settles
Keyword(s):  
High Speed ◽  
Fluid Dynamic ◽  
Human Subjects ◽  
Tracer Gas ◽  
Airborne Infection ◽  
Dynamic Mechanisms ◽  
Human Volunteers ◽  
Natural Mechanism ◽  
Time Changes ◽  
Aerosol Infection

Various infectious agents are known to be transmitted naturally via respiratory aerosols produced by infected patients. Such aerosols may be produced during normal activities by breathing, talking, coughing and sneezing. The schlieren optical method, previously applied mostly in engineering and physics, can be effectively used here to visualize airflows around human subjects in such indoor situations, non-intrusively and without the need for either tracer gas or airborne particles. It accomplishes this by rendering visible the optical phase gradients owing to real-time changes in air temperature. In this study, schlieren video records are obtained of human volunteers coughing with and without wearing standard surgical and N95 masks. The object is to characterize the exhaled airflows and evaluate the effect of these commonly used masks on the fluid-dynamic mechanisms that spread infection by coughing. Further, a high-speed schlieren video of a single cough is analysed by a computerized method of tracking individual turbulent eddies, demonstrating the non-intrusive velocimetry of the expelled airflow. Results show that human coughing projects a rapid turbulent jet into the surrounding air, but that wearing a surgical or N95 mask thwarts this natural mechanism of transmitting airborne infection, either by blocking the formation of the jet (N95 mask), or by redirecting it in a less harmful direction (surgical mask).

scholarly journals Lymphocyte metallothionein mRNA responds to marginal zinc intake in human volunteers

2000 ◽  
Vol 84 (5) ◽  
pp. 747-756 ◽  
Author(s):  
Adrian K. Allan ◽  
Gabrielle M. Hawksworth ◽  
Leslie R. Woodhouse ◽  
Barbara Sutherland ◽  
Janet C. King ◽  
...  
Keyword(s):  
Human Subjects ◽  
Baseline Period ◽  
Zn Deficiency ◽  
Pcr Assay ◽  
Laser Induced Fluorescence Detection ◽  
Pcr Product ◽  
Human Volunteers ◽  
Actin Mrna ◽  
Zn Status ◽  
Diagnostic Indicator

Marginal Zn deficiency is thought to be prevalent in both developed and developing countries. However, the extent of Zn deficiency is not known, due to the lack of a reliable diagnostic indicator. Blood plasma and erythrocyte concentrations of metallothionein (MT) reflect Zn status, but measurement of MT is dependent on the availability of sensitive immunoassays. Our aim was to show whether measurement of T lymphocyte MT-2A mRNA, using a competitive reverse transcriptase (RT)–polymerase chain reaction (PCR) assay, could indicate Zn status in human subjects in a residential Zn-depletion study. In the study, the Zn intake of seven volunteers was maintained at 13·7 mg/d for 5 weeks (baseline) followed by 4·6 mg/d for 10 weeks (marginal intake) and then 13·7 mg/d (repletion) for 5 weeks. The quantitative assay was developed using standard techniques and concentrations of MT-2A mRNA were normalized by reference to β-actin mRNA which was also measured by competitive RT–PCR assay. An alternative method of measuring the PCR product using capillary electrophoresis with laser-induced fluorescence detection was also evaluated. There was considerable inter-individual variation in MT-2A mRNA concentration and the mean level at the end of the baseline period was 10·3 (SE 3·7) fg MT-2A mRNA/pg β-actin mRNA, which then decreased by 64 % during the low Zn intake period. After repletion, MT-2A mRNA returned to baseline concentrations. In contrast, plasma Zn was unchanged by marginal Zn intake or repletion. The effect of low Zn in all individuals was consistent. We conclude that this assay is a sensitive method of evaluating marginal changes in dietary Zn intake.

scholarly journals Localization of Haemophilus ducreyi at the Pustular Stage of Disease in the Human Model of Infection

2000 ◽  
Vol 68 (4) ◽  
pp. 2309-2314 ◽  
Author(s):  
Margaret E. Bauer ◽  
Stanley M. Spinola
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Human Subjects ◽  
Haemophilus Ducreyi ◽  
Human Model ◽  
Stage Of Disease ◽  
The One ◽  
Secondary Antibodies

ABSTRACT To localize Haemophilus ducreyi in vivo, human subjects were experimentally infected with H. ducreyi until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformaldehyde, and cryosectioned. Sections were stained with polyclonal anti-H. ducreyi antiserum or H. ducreyi-specific monoclonal antibodies (MAbs) and fluorescently tagged secondary antibodies and examined by confocal microscopy. We identified H. ducreyi in 16 of 18 pustules but did not detect bacteria in the one papule examined. H. ducreyi was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demonstrated that H. ducreyi expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining with polyclonal anti-H. ducreyi antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules. We were unable to detect bacteria associated with keratinocytes in the samples examined. The data suggest that H. ducreyi is found primarily in association with neutrophils and in the dermis at the pustular stage of disease in the human model of infection.

scholarly journals A DltA Mutant of Haemophilus ducreyi Is Partially Attenuated in Its Ability To Cause Pustules in Human Volunteers

2006 ◽  
Vol 74 (2) ◽  
pp. 1394-1397 ◽  
Author(s):  
Diane Janowicz ◽  
Isabelle Leduc ◽  
Kate R. Fortney ◽  
Barry P. Katz ◽  
Christopher Elkins ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Human Serum ◽  
Parent Strain ◽  
Outer Membrane Proteins ◽  
The Other ◽  
Haemophilus Ducreyi ◽  
Serum Resistance ◽  
Human Volunteers ◽  
Serum Killing ◽  
Normal Human

ABSTRACT Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.

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