bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing the Risk of Primary Amoebic Meningoencephalitis from Swimming in the Presence of EnvironmentalNaegleria fowleri
Authors: Cabanes Pierre-André , Wallet France , Pringuez Emmanuelle , Pernin Pierre
Published: July 2002

Free-livingNaegleria fowleriamoebae cause primary amoebic meningoencephalitis (PAM). Because of the apparent conflict between their ubiquity and the rarity of cases observed, we sought to develop a model characterizing the risk of PAM after swimming as a function of the concentration ofN. fowleri. The probability of death from PAM as a function of the number of amoebae inhaled is modeled according to results obtained from animals infected with amoeba strains. The calculation of the probability of inhaling one or more amoebae while swimming is based on a double hypothesis: that the distribution of amoebae in the water follows a Poisson distribution and that the mean quantity of water inhaled while swimming is 10 ml. The risk of PAM for a given concentration of amoebae is then obtained by summing the following products: the probability of inhalingnamoebae × the probability of PAM associated with inhaling thesenamoebae. We chose the lognormal model to assess the risk of PAM because it yielded the best analysis of the studentized residuals. Nonetheless, the levels of risk thereby obtained cannot be applied to humans without correction, because they are substantially greater than those indicated by available epidemiologic data. The curve was thus adjusted by a factor calculated with the least-squares method. This provides the PAM risk in humans as a function of theN. fowlericoncentration in the river. For example, the risk is 8.5 × 10−8at a concentration of 10N. fowleriamoebae per liter.
Generation of a Novel
Saccharomyces cerevisiae
Strain That Exhibits Strong Maltose Utilization and Hyperosmotic Resistance Using Nonrecombinant Techniques
Authors: Higgins Vincent J. , Bell Philip J. L. , Dawes Ian W. , Attfield Paul V.
Published: July 2002

A yeast strain capable of leavening both unsugared and sweet bread dough efficiently would reduce the necessity of carrying out the expensive procedure of producing multiple baker's yeast strains. But issues involving the use of genetically modified foods have rendered the use of recombinant techniques for developing yeast strains controversial. Therefore, we used strong selection and screening systems in conjunction with traditional mass mating techniques to develop a strain of
Saccharomyces cerevisiae
that efficiently leavens both types of dough.
Intraspecies Genomic Groups in
Enterococcus faecium
and Their Correlation with Origin and Pathogenicity
Authors: Vancanneyt Marc , Lombardi Angiolella , Andrighetto Christian , Knijff Edo , Torriani Sandra , Björkroth K. Johanna , Franz Charles M. A. P. , Foulquié Moreno María R. , Revets Hilde , De Vuyst Luc , Swings Jean , Kersters Karel , Dellaglio Franco , Holzapfel Wilhelm H.
Published: July 2002

Seventy-eight
Enterococcus faecium
strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of
Sma
I restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of
E. faecium
strains was not confirmed.
Spatially Oscillating Activity and Microbial Succession of Mercury-Reducing Biofilms in a Technical-Scale Bioremediation System
Authors: von Canstein Harald , Li Ying , Leonhäuser Johannes , Haase Elke , Felske Andreas , Deckwer Wolf-Dieter , Wagner-Döbler Irene
Published: July 2002

Mercury-contaminated chemical wastewater of a mercury cell chloralkali plant was cleaned on site by a technical-scale bioremediation system. Microbial mercury reduction of soluble Hg(II) to precipitating Hg(0) decreased the mercury load of the wastewater during its flow through the bioremediation system by up to 99%. The system consisted of a packed-bed bioreactor, where most of the wastewater's mercury load was retained, and an activated carbon filter, where residual mercury was removed from the bioreactor effluent by both physical adsorption and biological reduction. In response to the oscillation of the mercury concentration in the bioreactor inflow, the zone of maximum mercury reduction oscillated regularly between the lower and the upper bioreactor horizons or the carbon filter. At low mercury concentrations, maximum mercury reduction occurred near the inflow at the bottom of the bioreactor. At high concentrations, the zone of maximum activity moved to the upper horizons. The composition of the bioreactor and carbon filter biofilms was investigated by 16S-23S ribosomal DNA intergenic spacer polymorphism analysis. Analysis of spatial biofilm variation showed an increasing microbial diversity along a gradient of decreasing mercury concentrations. Temporal analysis of the bioreactor community revealed a stable abundance of two prevalent strains and a succession of several invading mercury-resistant strains which was driven by the selection pressure of high mercury concentrations. In the activated carbon filter, a lower selection pressure permitted a steady increase in diversity during 240 days of operation and the establishment of one mercury-sensitive invader.
Inactivation of the Regulatory Gene
algU
or
gacA
Can Affect the Ability of Biocontrol
Pseudomonas fluorescens
CHA0 To Persist as Culturable Cells in Nonsterile Soil
Authors: Mascher Fabio , Moënne-Loccoz Yvan , Schnider-Keel Ursula , Keel Christoph , Haas Dieter , Défago Geneviève
Published: July 2002

Rifampin-resistant
Pseudomonas fluorescens
CHA0-Rif and mutants in which the regulatory gene
algU
(encoding sigma factor σ
E
) or
gacA
(encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional
algU
and (particularly)
gacA
were needed for CHA0-Rif to maintain cell culturability in soil.
Assessment of the Genetic Diversity among Arcobacters Isolated from Poultry Products by Using Two PCR-Based Typing Methods
Authors: Houf Kurt , De Zutter Lieven , Van Hoof Jan , Vandamme Peter
Published: July 2002

In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of
Arcobacter butzleri
,
Arcobacter cryaerophilus
, and
Arcobacter skirrowii
. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of
Arcobacter
isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as
A. butzleri
(
n
= 182) and
A. cryaerophilus
(
n
= 46). A total of 131 types (91
A. butzleri
types and 40
A. cryaerophilus
types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.
Microbial Load of Drinking Water Reservoir Tributaries during Extreme Rainfall and Runoff
Authors: Kistemann T. , Claßen T. , Koch C. , Dangendorf F. , Fischeder R. , Gebel J. , Vacata V. , Exner M.
Published: July 2002

Hygienic and microbiological examinations of watercourses are usually not carried out during heavy rainfall and runoff events. After rainfall or snowmelt, there are often massive increases in turbidity in flooding creeks in mountain ranges, which are frequently interpreted as an indication of microbial contamination. The aim of this study was to quantify the microbial loads of watercourses during such runoff events and to compare these loads with loads occurring during regular conditions. In a 14-month monitoring period we investigated the microbial loads of three tributaries of different drinking water reservoirs. A total of 99 water samples were taken under different runoff conditions and analyzed to determine physical, chemical, bacterial, and parasitic parameters. Thirty-two water samples were considered event samples during nine measuring series. The criteria for events, based on duration and intensity of precipitation, water depth gauge measurements, and dynamics, had been fixed before the investigation for each creek individually. Of the physical and chemical parameters examined, only the turbidity, pH, and nitrate values differed clearly from the values obtained for regular samples. Most of the bacteriological parameters investigated (colony,
Escherichia coli
, coliform, fecal streptococcal, and
Clostridium perfringens
counts) increased considerably during extreme runoff events. If relevant sources of parasitic contamination occurred in catchment areas, the concentrations of
Giardia
and
Cryptosporidium
rose significantly during events. The results show that substantial shares of the total microbial loads in watercourses and in drinking water reservoirs result from rainfall and extreme runoff events. Consequently, regular samples are considered inadequate for representing the microbial contamination of watercourse systems. The procedures for raw water surveillance in the context of multiple-barrier protection and risk assessment ought to include sampling during extreme runoff situations.
Differences in Growth of
Salmonella enterica
and
Escherichia coli
O157:H7 on Alfalfa Sprouts
Authors: Charkowski A. O. , Barak J. D. , Sarreal C. Z. , Mandrell R. E.
Published: July 2002

Sprout producers have recently been faced with several
Salmonella enterica
and
Escherichia coli
O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with
S. enterica
and
E. coli
O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds.
S. enterica
strains grew an average of 3.7 log
10
on sprouting seeds over 2 days, while
E. coli
O157:H7 strains grew significantly less, an average of 2.3 log
10
. The initial
S. enterica
or
E. coli
O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both
S. enterica
and
E. coli
O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of
E. coli
O157:H7. Colonization of sprouting alfalfa seeds by
S. enterica
serovar Newport and
E. coli
O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy.
Salmonella
serovar Newport colonized both seed coats and sprout roots as aggregates, while
E. coli
O157:H7 colonized only sprout roots.
Uranium Reduction by
Desulfovibrio desulfuricans
Strain G20 and a Cytochrome
c
3
Mutant
Authors: Payne Rayford B. , Gentry Darren M. , Rapp-Giles Barbara J. , Casalot Laurence , Wall Judy D.
Published: July 2002

Previous in vitro experiments with
Desulfovibrio vulgaris
strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome
c
3
could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome
c
3
mutant of
D. desulfuricans
strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome
c
3
appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.
Characterization of a Predominant Immunogenic Outer Membrane Protein of
Riemerella anatipestifer
Authors: Subramaniam Sumathi , Huang Bin , Loh Hilda , Kwang Jimmy , Tan Hai-Meng , Chua Kim-Lee , Frey Joachim
Published: July 2002

The
ompA
gene, encoding the 42-kDa major antigenic outer membrane protein OmpA of
Riemerella anatipestifer
, the etiololgical agent of septicemia anserum exsudativa, was cloned and expressed in
Escherichia coli
. Recombinant OmpA displayed a molecular mass similar to that predicted from the nucleotide sequence of the
ompA
gene but lower than that observed in total cell lysates of
R. anatipestifer
. The
ompA
gene showed a conserved C-terminal region comprising the OmpA-like domain and a variable N-terminal region. This structure is similar to those of the analogous outer membrane proteins of several gram-negative bacteria. However, OmpA of
R. anatipestifer
contains six EF-hand calcium-binding domains and two PEST regions, which distinguish it from other outer membrane proteins. The occurrence of these motifs in OmpA suggests a possible role in virulence for this protein. The
ompA
gene is present in the
R. anatipestifer
type strain and in all serotype reference strains. However, it exhibits some minor genetic heterogeneity among different serotypes, which seems not to affect the strong antigenic characteristics of the protein. OmpA is a conserved and strong antigenic determinant of
R. anatipestifer
and hence is suggested to be a valuable protein for the serodetection of
R. anatipestifer
infections, independent of their serotype.
Description of a Nonlethal Herpes Simplex Virus Type 1 Glycoprotein D Deletion Mutant Affecting a Site Frequently Used for PCR
Authors: Coyle P. V. , Jain S. , Wyatt D. , McCaughey C. , O'Neill H. J.
Published: July 2002

We report a herpes simplex virus type 1 mutant which failed to amplify with a commonly used glycoprotein D primer set. The virus contained a nine-base deletion in the gene's 5′ nontranslated region. The altered amplicon was clearly distinguishable on a 4% high-resolution agarose gel.
Analysis of Complement Fixation and Commercial Enzyme Immunoassays for Detection of Antibodies to
Mycoplasma pneumoniae
in Human Serum
Authors: Thacker W. Lanier , Talkington Deborah F.
Published: July 2002

The Meridian ImmunoCard (IC), GenBio ImmunoWELL-IgM, and Remel EIA commercial antibody tests are qualitative enzyme immunoassays that detect antibodies to
Mycoplasma pneumoniae
in serum. These tests were compared to an
M. pneumoniae
complement fixation (CF) assay, which uses a commercially available antigen component. The Meridian IC and the ImmunoWELL-IgM detect immunoglobulin M (IgM) only; the Remel EIA and the CF test detect both IgM and IgG antibodies. Detection of specific IgM antibody, which appears early in infection, can be, but is not always, indicative of a recent or current infection. Paired serum samples from 64 adult patients with probable
M. pneumoniae
infection were examined with each of the four tests. Thirty (47%) of the 64 acute-phase sera were IgM positive by Meridian IC, 26 (41%) were positive by Remel EIA, 24 (38%) were positive by CF, and 15 (23%) were positive by ImmunoWELL-IgM. When both the acute- and convalescent-phase serum samples from each patient were examined, 61 (95%) of the 64 patients were positive by CF, 60 patients (94%) were positive by Remel EIA, 52 patients (81%) were IgM positive by the Meridian IC, and 29 patients (45%) were IgM positive by the ImmunoWELL-IgM assay. The Meridian IC was more sensitive than the other tests for early detection of IgM antibodies. However, after examining paired serum samples, we concluded that the detection of IgM alone may not be useful for all cases of mycoplasma infection, especially in an adult population.
Structural and Immunological Characteristics of a 28-Kilodalton Cruzipain-Like Cysteine Protease ofParagonimus westermaniExpressed in the Definitive Host Stage
Authors: Yun Doo-Hee , Chung Joon-Yong , Chung Young-Bae , Bahk Young-Yil , Kang Shin-Yong , Kong Yoon , Cho Seung-Yull
Published: July 2002

A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adultParagonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed inEscherichia coliwas used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.
Cell-Mediated Immune Response to Tuberculosis Antigens: Comparison of Skin Testing and Measurement of In Vitro Gamma Interferon Production in Whole-Blood Culture
Authors: Katial Rohit K. , Hershey Joyce , Purohit-Seth Tejashiri , Belisle John T. , Brennan Patrick J. , Spencer John S. , Engler Renata J. M.
Published: July 2002

Although delayed-type hypersensitivity skin testing with tuberculin purified protein derivative (PPD) is the standard for tuberculosis screening, its variability suggests the need for a more sensitive, noninvasive test. An in vitro whole-blood assay has been proposed as an alternative. Using health care worker volunteers, we confirmed the correlation between PPD skin test (PPD-ST) results (positive, induration of >15 mm) and a standardized gamma interferon (IFN-γ) assay, QuantiFERON-TB (Q-IFN), manufactured by CSL Biosciences in Australia, and we evaluatedMycobacterium tuberculosisculture subfractions as potential substitutes for PPD. Twenty healthy volunteers with positive PPD-ST results and 20 PPD-ST-negative controls were enrolled. Whole blood was cultured with human PPD antigens (HuPPD),Mycobacterium aviumcomplex (MAC) PPD, phytohemagglutinin (PHA), and fourM. tuberculosisculture subfractions: low-molecular-weight culture, filtrate, culture filtrate without lipoarabinomannan, soluble cell wall proteins, and cytosolic proteins, all developed fromM. tuberculosisstrain H37RV. Secretion of IFN-γ (expressed as international units per milliliter) was measured by an enzyme immunoassay. The PPD or subculture fraction response as a percentage of the PHA response was used to determine positivity. Sixteen of 20 PPD-ST-positive individuals were classified asM. tuberculosispositive by Q-IFN, and 1 was classified as MAC positive. Sixteen of 20 PPD-ST-negative individuals wereM. tuberculosisnegative by Q-IFN, 2 were MAC positive, and 2 wereM. tuberculosispositive. The tuberculosis culture subfractions stimulated IFN-γ production in PPD-ST-positive volunteers, and significant differences could be seen between the two PPD-ST groups with all subfractions except soluble cell wall protein; however, the response was variable and no better than the Q-IFN PPD. The agreement between the Q-IFN test and the PPD-ST was good (Cohen's kappa = 0.73). The Q-IFN assay can be a useful tool in further studies of immune responses toM. tuberculosisantigens.
Two Methods for Rapid Serological Diagnosis of Acute Leptospirosis
Authors: Levett Paul N. , Branch Songee L. , Whittington Carol U. , Edwards Charles N. , Paxton Helene
Published: July 2002

Leptospirosis is a common and underdiagnosed zoonosis. Two rapid assays for serological diagnosis of acute leptospirosis in diagnostic laboratories, the immunoglobulin M (IgM)-dipstick assay and the indirect hemagglutination assay (IHA), were evaluated and compared with standard assays. Sera were examined from 104 patients admitted to a hospital for investigation in a leptospirosis diagnostic protocol. Specimens for serology were taken on days 1 and 4 of the patients' hospital stay. Antibodies were detected using an IgM-enzyme-linked immunosorbent assay (ELISA), microscopic agglutination test (MAT), an IgM-dipstick assay, and an IHA. Fifty-one patients were found to have leptospirosis. The sensitivity of the IgM-dipstick assay was 98%, its specificity was 90.6%, its positive predictive value was 90.9%, and its negative predictive value was 98%. The sensitivity of the IHA was 92.2%, its specificity was 94.4%, its positive predictive value was 95.9%, and its negative predictive value was 92.7%. The standard IgM-ELISA and MAT, were positive in the first samples tested from 67 and 55% of the cases, respectively, and the rapid IgM-dipstick assay and IHA were positive in 71 and 49%, respectively, in the first sample tested. Both rapid assays are highly sensitive and specific. Neither requires specialized equipment, and both are suitable for use in diagnostic laboratories.
Suppression of Endotoxin- and Staphylococcal Exotoxin-Induced Cytokines and Chemokines by a Phospholipase C Inhibitor in Human Peripheral Blood Mononuclear Cells
Authors: Krakauer Teresa
Published: July 2002

Excessive release of proinflammatory cytokines from cells stimulated with lipopolysaccharide (LPS) or staphylococcal exotoxin (SE) mediates the pathophysiologic manifestations of septic shock. Tricyclodecan-9-yl (D609), an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed LPS- or SE-induced cytokines and chemokines in human peripheral blood mononuclear cells. These data suggest a potential role for D609 in the treatment of septic shock.
Celiac Disease-Associated Autoimmune Endocrinopathies
Authors: Kumar Vijay , Rajadhyaksha Manoj , Wortsman Jacobo
Published: July 2002

Celiac disease (CD) is an autoimmune disorder induced by gluten intake in genetically susceptible individuals. It is characterized by the presence of serum antibodies to endomysium, reticulin, gliadin, and tissue transglutaminase. The incidence of CD in various autoimmune disorders is increased 10- to 30-fold in comparison to the general population, although in many cases CD is clinically asymptomatic or silent. The identification of such cases with CD is important since it may help in the control of type I diabetes or endocrine functions in general, as well as in the prevention of long-term complications of CD, such as lymphoma. It is believed that CD may predispose an individual to other autoimmune disorders such as type I diabetes, autoimmune thyroid, and other endocrine diseases and that gluten may be a possible trigger. The onset of type I diabetes at an early age in patients with CD, compared to non-CD, and the prevention or delay in onset of diabetes by gluten-free diet in genetically predisposed individuals substantiates this antigen trigger hypothesis. Early identification of CD patients in highly susceptible population may result in the treatment of subclinical CD and improved control of associated disorders.
Enzyme-Linked Immunospot Assays Provide a Sensitive Tool for Detection of Cytokine Secretion by Monocytes
Authors: Kouwenhoven Mathilde , Özenci Volkan , Teleshova Natalia , Hussein Yassir , Huang Yu-Min , Eusebio Alexandre , Link Hans
Published: July 2002

Blood monocytes as well as tissue-differentiated macrophages play a pivotal role in controlling immune reactions. Monocytes regulate the extent, nature, and duration of immune responses by secretion of cytokines. Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-10, and IL-12 are of particular interest, since IL-12 shifts the immune response towards a Th1 type, facilitating the production of, e.g., TNF-α and IL-6, while IL-10 counteracts Th1 responses and promotes the production of Th2-related cytokines such as IL-4. A tight regulation of these four cytokines keeps the balance and decides whether Th1 or Th2 will predominate in immune reactions. Enzyme-linked immunospot (ELISPOT) assays are among the most-sensitive and -specific methods available for cytokine research. They permit ex vivo identification of individual cells actively secreting cytokines. In the present study we prepared monocytes from healthy subjects' blood and adapted ELISPOT assays to define optimal conditions to detect and enumerate monocytes secreting IL-6, TNF-α, IL-10, and IL-12. The optimal time for monocyte incubation was 24 h, and optimal monocyte numbers (in cells per well) were 2,000 for IL-6, 1,000 for TNF-α, 50,000 for IL-10, and 100,000 for enumeration of IL-12 secreting monocytes. Among healthy subjects, 10% ± 5% of the monocytes secreted IL-6, 12% ± 12% secreted TNF-α, 0.1% ± 0.1% secreted IL-10, and 0.2% ± 0.3% secreted IL-12 (values are means ± standard deviations). In conclusion, ELISPOT assays constitute a valuable tool to enumerate monocytes secreting IL-6, TNF-α, IL-10, and IL-12 and probably to enumerate monocytes secreting other cytokines and proteins.
Immunobiological Effects of Fumonisin B1 in Experimental Subchronic Mycotoxicoses in Rats
Authors: Theumer M. G. , López A. G. , Masih D. T. , Chulze S. N. , Rubinstein H. R.
Published: July 2002

Fumonisin B1 (FB1), the principal secondary metabolite produced by the fungusFusarium verticillioides(Gibberella fujikuroimating population A), is a potent toxin that can be found in fungus-contaminated corn and corn-based food products. We have investigated the immunobiological effects of subchronic dietary exposure to FB1 in male Wistar rats. Animals were fed with diets containing 0 (control) or 100 ppm of FB1 for 12 weeks. The total FB1 intake on day 90 was 810 mg/kg of body weight. Food consumption, body weight, and body weight gain on day 90 were reduced in animals exposed to FB1. Histopathologic changes consisted of histiocytic perivascular infiltrate and an increased number of Kupffer cells in the liver, necrosis and apoptosis of tubular epithelial cells in the kidney, and increased mitotic figures and lymphocytic infiltrate in the small intestine. Serum enzyme alkaline phosphatase was significantly elevated in rats fed FB1, while triglyceride levels decreased compared to controls. Treatment with FB1 in vivo or in vitro did not have a significant effect on mitogen-induced proliferation of spleen mononuclear cells. However, increased levels of interleukin-4 (IL-4) and decreased levels of IL-10 were released by these cells in culture compared to controls. FB1 in vivo or in vitro decreased the hydrogen peroxide (H2O2) released by peritoneal macrophages, while no changes in levels of superoxide anion produced by total peritoneal cells were detected. The results from the present work demonstrate that subchronic FB1 intake could affect the small intestine and alter the interleukin profile and some main functions of macrophages in antitumor activity.
Genotypic Testing for Human Immunodeficiency Virus Type 1 Drug Resistance
Authors: Shafer Robert W.
Published: July 2002

There are 16 approved human immunodeficiency virus type 1 (HIV-1) drugs belonging to three mechanistic classes: protease inhibitors, nucleoside and nucleotide reverse transcriptase (RT) inhibitors, and nonnucleoside RT inhibitors. HIV-1 resistance to these drugs is caused by mutations in the protease and RT enzymes, the molecular targets of these drugs. Drug resistance mutations arise most often in treated individuals, resulting from selective drug pressure in the presence of incompletely suppressed virus replication. HIV-1 isolates with drug resistance mutations, however, may also be transmitted to newly infected individuals. Three expert panels have recommended that HIV-1 protease and RT susceptibility testing should be used to help select HIV drug therapy. Although genotypic testing is more complex than typical antimicrobial susceptibility tests, there is a rich literature supporting the prognostic value of HIV-1 protease and RT mutations. This review describes the genetic mechanisms of HIV-1 drug resistance and summarizes published data linking individual RT and protease mutations to in vitro and in vivo resistance to the currently available HIV drugs.
Innate Immunity toMycobacterium tuberculosis
Authors: van Crevel Reinout , Ottenhoff Tom H. M. , van der Meer Jos W. M.
Published: July 2002

The different manifestations of infection with Mycobacterium tuberculosis reflect the balance between the bacillus and host defense mechanisms. Traditionally, protective immunity to tuberculosis has been ascribed to T-cell-mediated immunity, with CD4+T cells playing a crucial role. Recent immunological and genetic studies support the long-standing notion that innate immunity is also relevant in tuberculosis. In this review, emphasis is on these natural, innate host defense mechanisms, referring to experimental data (e.g., studies in gene knockout mice) and epidemiological, immunological, and genetic studies in human tuberculosis. The first step in the innate host defense is cellular uptake of M. tuberculosis, which involves different cellular receptors and humoral factors. Toll-like receptors seem to play a crucial role in immune recognition of M. tuberculosis, which is the next step. The subsequent inflammatory response is regulated by production of pro- and anti-inflammatory cytokines and chemokines. Different natural effector mechanisms for killing of M. tuberculosis have now been identified. Finally, the innate host response is necessary for induction of adaptive immunity to M. tuberculosis. These basic mechanisms augment our understanding of disease pathogenesis and clinical course and will be of help in designing adjunctive treatment strategies.
Mycobacterium bovisBCG Induces Similar Immune Responses and Protection by Rectal and Parenteral Immunization Routes
Authors: M Abolhassani , Lagranderie M. , Chavarot P. , Balazuc A.-M. , Marchal G.
Published: July 2002

We compared cellular immune responses to rectal, subcutaneous, and intradermal administration ofMycobacterium bovisBCG for 5 to 20 weeks in mice, guinea pigs, and macaques. Strong lymphoproliferative responses were induced in spleen cells after in vitro stimulation with purified protein derivative in guinea pigs and macaques, whatever the route of immunization. Comparable high numbers of gamma interferon- and tumor necrosis factor alpha-producing cells were found in the spleen after rectal, subcutaneous, and intradermal immunization of mice and macaques. Similar levels of precursors of cytotoxic T lymphocytes specific for mycobacterial antigens were observed in mice for all immunization routes. In macaques, cytotoxic activity, determined only at the end of the experiment (20 weeks), was similar after rectal and intradermal immunization. Six months after immunization, rectal and subcutaneous routes induced in mice similar levels of protective immunity against challenge with a virulentMycobacterium tuberculosisstrain (H37Rv). Rectal immunization gave immune responses and protective capacity similar to those for parenteral immunization and seemed to be a promising new route of vaccination against tuberculosis; in our study, immunization via the rectal route never induced side effects associated with parenteral routes (axillary adenitis) and could also effectively reduce the risks of viral transmission associated with unsafe injections in the developing world.
Peritoneal Culture Alters
Streptococcus pneumoniae
Protein Profiles and Virulence Properties
Authors: Orihuela Carlos J. , Janssen Rob , Robb Christopher W. , Watson David A. , Niesel David W.
Published: July 2002

We have examined the properties of
Streptococcus pneumoniae
cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in
ply
expression and a 2.2-fold increase in
capA3
expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.
imp
A, a Gene Coding for an Inner Membrane Protein, Influences Colonial Morphology of
Actinobacillus actinomycetemcomitans
Authors: Mintz Keith P. , Fives-Taylor Paula M.
Published: July 2002

Directed mutagenesis of a gene coding for a membrane protein of the periodontopathogen
Actinobacillus actinomycetemcomitans
was achieved by conjugation. The gene was disrupted by insertion of an antibiotic cassette into a unique endonuclease restriction sequence engineered by inverse PCR. The disrupted gene was cloned into a conjugative plasmid and transferred from
Escherichia coli
to
A. actinomycetemcomitans
. The allelic replacement mutation resulted in the loss of a 22-kDa inner membrane protein. The loss of this protein (ImpA) resulted in changes in the outer membrane protein composition of the bacterium. Concurrent with the mutation in
impA
was a change in the pattern of growth of the mutant bacteria in broth cultures. The progenitor bacteria grew as a homogeneous suspension of cells compared to a granular, autoaggregating adherent cell population described for the mutant bacteria. These data suggest that ImpA may play a regulatory role or be directly involved in protein(s) that are exported and associated with colony variations in
A. actinomycetemcomitans.
Sialylation of Lipooligosaccharide Cores Affects Immunogenicity and Serum Resistance of
Campylobacter jejuni
Authors: Guerry Patricia , Ewing Cheryl P. , Hickey Thomas E. , Prendergast Martina M. , Moran Anthony P.
Published: July 2002

Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of
Campylobacter jejuni
MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM
2
ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (
cstII
) and a putative
N
-acetylmannosamine synthetase (
neuC1
), part of the biosynthetic pathway of
N
-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of
C. jejuni
MSC57360. Loss of sialic acid in the core of the
neuC1
mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative β-1,4-
N
-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of
N
-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the
cgt
mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.
Differential Alteration in Intestinal Epithelial Cell Expression of Toll-Like Receptor 3 (TLR3) and TLR4 in Inflammatory Bowel Disease
Authors: Cario Elke , Podolsky Daniel K.
Published: July 2002

Initiation and perpetuation of the inflammatory intestinal responses in inflammatory bowel disease (IBD) may result from an exaggerated host defense reaction of the intestinal epithelium to endogenous lumenal bacterial flora. Intestinal epithelial cell lines constitutively express several functional Toll-like receptors (TLRs) which appear to be key regulators of the innate response system. The aim of this study was to characterize the expression pattern of TLR2, TLR3, TLR4, and TLR5 in primary intestinal epithelial cells from patients with IBD. Small intestinal and colonic biopsy specimens were collected from patients with IBD (Crohn's disease [CD], ulcerative colitis [UC]) and controls. Non-IBD specimens were assessed by immunofluorescence histochemistry using polyclonal antibodies specific for TLR2, TLR3, TLR4, and TLR5. Primary intestinal epithelial cells (IEC) of normal mucosa constitutively expressed TLR3 and TLR5, while TLR2 and TLR4 were only barely detectable. In active IBD, the expression of TLR3 and TLR4 was differentially modulated in the intestinal epithelium. TLR3 was significantly downregulated in IEC in active CD but not in UC. In contrast, TLR4 was strongly upregulated in both UC and CD. TLR2 and TLR5 expression remained unchanged in IBD. These data suggest that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium, implying that alterations in the innate response system may contribute to the pathogenesis of these disorders.
Evaluation of the Virulence of a
Streptococcus pneumoniae
Neuraminidase-Deficient Mutant in Nasopharyngeal Colonization and Development of Otitis Media in the Chinchilla Model
Authors: Tong H. H. , Blue L. E. , James M. A. , DeMaria T. F.
Published: July 2002

Considerable evidence has implicated
Streptococcus pneumoniae
neuraminidase in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, an
S. pneumoniae
neuraminidase-deficient mutant, ΔNA1, has been constructed by insertion-duplication mutagenesis of the
nanA
gene of
S. pneumoniae
strain D39. The relative ability of ΔNA1 and the D39 parent strain to colonize the nasopharynx and to induce OM subsequent to intranasal inoculation and to survive in the middle ear cleft after direct challenge of the middle ear were evaluated in the chinchilla model. Nasopharyngeal colonization data indicate a significant difference in the ability of the ΔNA1 mutant to colonize as well as to persist in the nasopharynx. The neuraminidase-deficient mutant was eliminated from the nasopharynx 2 weeks earlier than the D39 parent strain. Both the parent and the mutant exhibited similar virulence levels and kinetics during the first week after direct inoculation of the middle ear. The ΔNA1 neuraminidase-deficient mutant, however, was then completely eliminated from the middle ear by day 10 postchallenge, 11 days before the D39 parent strain. Data from this study indicate that products of the
nanA
gene have an impact on the ability of
S. pneumoniae
to colonize and persist in the nasopharynx as well as the middle ear.
Capsular Polysaccharide Is a Major Complement Resistance Factor in Lipopolysaccharide O Side Chain-Deficient
Klebsiella pneumoniae
Clinical Isolates
Authors: Álvarez Dolores , Merino Susana , Tomás Juan M. , Benedí Vicente J. , Albertí Sebastián
Published: July 2002

We have previously demonstrated the existence of
Klebsiella pneumoniae
clinical isolates deficient in the lipopolysaccharide O side chain, the major factor for resistance to complement-mediated killing in this bacterial species. These isolates are complement resistant, and their mechanisms to resist complement were investigated by selecting transposon-generated complement-sensitive mutants. One mutant with a drastically reduced capacity to grow in nonimmune human serum carried the transposon inserted in an open reading frame of a gene cluster involved in capsule synthesis. This mutant produced less capsule, bound more molecules of the complement component C3, and was more sensitive to complement-mediated and opsonophagocytic killings than was the parent strain. Four additional clinical isolates representing four different K serotypes were studied, and results showed that capsular polysaccharide is a major complement resistance factor in these O side chain-deficient isolates.
Borrelia burgdorferiGene Expression In Vivo and Spirochete Pathogenicity
Authors: Anguita Juan , Samanta Swapna , Revilla Beatriz , Suk Kyoungho , Das Subrata , Barthold Stephen W. , Fikrig Erol
Published: July 2002

Borrelia burgdorferispirochetes that do not cause arthritis or carditis were developed and used to investigate Lyme disease pathogenesis. A clonal isolate ofB. burgdorferiN40 (cN40), which induces disease in C3H/HeN (C3H) mice, was repeatedly passaged in vitro to generate nonpathogenic spirochetes. The passage 75 isolate (N40-75) was infectious for C3H mice but did not cause arthritis or carditis, and spirochetes were at low levels or absent in the joints or hearts, respectively. N40-75 could, however, cause disease in severe combined immunodeficient (SCID) mice, suggesting that the response in immunocompetent mice prevented effective spirochete dissemination and the subsequent development of arthritis and carditis. Administration of immune sera at 4 days after spirochete challenge aborted N40-75, but not cN40, infection in SCID mice. AB. burgdorferigenomic expression library was differentially probed with sera from cN40- and N40-75-infected mice, to identify genes that may not be effectively expressed by N40-75 in vivo. N40-75 was defective in the up-regulation of several genes that are preferentially expressed during mammalian infection, includingdbpAB,bba64, and genes that map to the cp32 family of plasmids. These data suggest that adaptation and gene expression may be required forB. burgdorferito effectively colonize the host, evade humoral responses, and cause disease.
Activation of Intercellular Adhesion Molecule 1 Expression byHelicobacter pyloriIs Regulated by NF-κB in Gastric Epithelial Cancer Cells
Authors: Mori Naoki , Wada Akihiro , Hirayama Toshiya , Parks Thomas P. , Stratowa Christian , Yamamoto Naoki
Published: July 2002

Interactions between leukocytes and epithelial cells may play a key role inHelicobacter pylori-associated gastric mucosal inflammation. This process is mediated by various cell adhesion molecules. The present study examined the molecular mechanisms leading toH. pylori-induced epithelial cell intercellular adhesion molecule-1 (ICAM-1; also called CD54) expression. Coculture of epithelial cells with cytotoxin-associated gene pathogenicity island-positive (cagPAI+)H. pyloristrains, but not with acagPAI−strain orH. pyloriculture supernatants, resulted in upregulation of steady-state mRNA levels and cell surface expression of ICAM-1. Coculture withH. pyloriinduced an increase in luciferase activity in cells which were transfected with a luciferase reporter gene linked to the 5′-flanking region of the ICAM-1 gene.H. pyloriactivated the ICAM-1 promoter via the NF-κB binding site. An inducible nuclear protein complex bound to the ICAM-1 NF-κB site and was identified as the NF-κB p50–p65 heterodimer.H. pyloriinduced the degradation of IκB-α, a major cytoplasmic inhibitor of NF-κB, and stimulated the expression of IκB-α mRNA. Pretreatment of epithelial cells with pyrrolidine dithiocarbamate, which blocks NF-κB activation, inhibitedH. pylori-induced ICAM-1 expression. THP-1 macrophagic cells, peripheral blood mononuclear cells, and purified neutrophils adhered toH. pylori-infected epithelial cells to a greater extent than to uninfected cells. These results show thatH. pyloridirectly induces expression of ICAM-1 on gastric epithelial cells in an NF-κB-dependent manner that may support leukocyte attachment during inflammation.
Specificity of Human Antibodies Reactive with Pneumococcal C Polysaccharide
Authors: Frasch Carl E. , Concepcion Nelydia F.
Published: July 2002

Antibodies reactive with C polysaccharide (PS) were found in healthy adults, pneumococcal patients, and vaccinees. These antibodies were not directed to the phosphocholine determinant of the C PS, as they appear to be in mice, since the human antibodies were inhibitable only with C PS. We found another population of phosphocholine-specific antibodies inhibitable only by phosphocholine and related compounds.
Hydroxamate Siderophores of
Histoplasma capsulatum
Authors: Howard Dexter H. , Rafie Ramin , Tiwari Anupama , Faull Kym F.
Published: July 2002

The zoopathogenic fungus
Histoplasma capsulatum
, like other eukaryotic aerobic microorganisms, requires iron for growth. Under conditions of low iron availability, the fungus secretes hydroxamates that function as siderophores (iron chelators). The experiments to be reported were designed to gather further information on the hydroxamate siderophores of
H. capsulatum
. The fungus was grown in a synthetic medium deferrated with the cationic exchange resin Chelex 100. Siderophores were detected after 4 days of incubation at 37°C in media containing 0.3 to 1.0 μM iron. The secretion was suppressed by 10 μM iron. The hydroxamates were purified by reverse-phase and size-exclusion chromatography. On the basis of ions observed during electrospray mass spectroscopy, five hydroxamate siderophores were tentatively identified: dimerum acid, acetyl dimerum acid, coprogen B, methyl coprogen B, and fusarinine (monomeric). A polyclonal antibody to dimerum acid was generated. This reagent cross-reacted with coprogen B and fusarinine. Thus, the antibody detects hydroxamates in all three families of siderophores excreted by
H. capsulatum
.
Ultrastructural Analysis of Developmental Events in
Chlamydia pneumoniae
-Infected Cells
Authors: Wolf Katerina , Fischer Elizabeth , Hackstadt Ted
Published: July 2002

Chlamydia pneumoniae
is an obligate intracellular parasite with a developmental cycle believed to be common to all members of the genus
Chlamydia
. We present a detailed description based on transmission and scanning electron microscopy of temporal events and inclusion structures throughout the
C. pneumoniae
AR-39 developmental cycle.