bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiologically Based Pharmacokinetic Model for Terbinafine in Rats and Humans
Authors: Hosseini-Yeganeh Mahboubeh , McLachlan Andrew J.
Published: July 2002

The aim of this study was to develop a physiologically based pharmacokinetic (PB-PK) model capable of describing and predicting terbinafine concentrations in plasma and tissues in rats and humans. A PB-PK model consisting of 12 tissue and 2 blood compartments was developed using concentration-time data for tissues from rats (n= 33) after intravenous bolus administration of terbinafine (6 mg/kg of body weight). It was assumed that all tissues except skin and testis tissues were well-stirred compartments with perfusion rate limitations. The uptake of terbinafine into skin and testis tissues was described by a PB-PK model which incorporates a membrane permeability rate limitation. The concentration-time data for terbinafine in human plasma and tissues were predicted by use of a scaled-up PB-PK model, which took oral absorption into consideration. The predictions obtained from the global PB-PK model for the concentration-time profile of terbinafine in human plasma and tissues were in close agreement with the observed concentration data for rats. The scaled-up PB-PK model provided an excellent prediction of published terbinafine concentration-time data obtained after the administration of single and multiple oral doses in humans. The estimated volume of distribution at steady state (Vss) obtained from the PB-PK model agreed with the reported value of 11 liters/kg. The apparent volume of distribution of terbinafine in skin and adipose tissues accounted for 41 and 52%, respectively, of theVssfor humans, indicating that uptake into and redistribution from these tissues dominate the pharmacokinetic profile of terbinafine. The PB-PK model developed in this study was capable of accurately predicting the plasma and tissue terbinafine concentrations in both rats and humans and provides insight into the physiological factors that determine terbinafine disposition.
Pharmacodynamic Effects of Nitroimidazoles Alone and in Combination with Clarithromycin on
Helicobacter pylori
Authors: Svensson Margareta , Ström Magnus , Nilsson Maud , Sörberg Mikael , Nilsson Lennart E.
Published: July 2002

Pharmacodynamic studies of
Helicobacter pylori
exposed to metronidazole and tinidazole alone and in combination with clarithromycin were performed by bioluminescence assay of intracellular ATP. The pharmacodynamic parameter control-related effective regrowth time (CERT) was used. CERT is defined as the time required for the resumption of logarithmic growth and a return of the level of growth to the preexposure inoculum in the test culture minus the corresponding time in the control culture. CERT measures the combined effects of the initial level of killing and postantibiotic effect. The incubation times and drug concentrations were chosen according to their half-lives and their clinically achievable concentrations. The study shows that the parameter CERT is useful for the testing of antibiotic combinations. The CERTs induced by clarithromycin, metronidazole, and tinidazole alone and in the combinations tested were concentration dependent, with no maximum response, indicating that the use of high doses may be preferable. The combinations with the highest concentrations induced synergistic effects and prevented regrowth. The use of tinidazole in combination with clarithromycin proved to give the longest CERTs, indicating that this is the most effective combination.
Novel Plasmid-Encoded Class C β-Lactamase (MOX-2) in
Klebsiella pneumoniae
from Greece
Authors: Raskine Laurent , Borrel Isabelle , Barnaud Guilène , Boyer Sophie , Hanau-Berçot Béatrice , Gravisse Jérome , Labia Roger , Arlet Guillaume , Sanson-Le-Pors Marie-José
Published: July 2002

Klebsiella pneumoniae
KOL, a clinical strain resistant to various β-lactams, was isolated from the stools of a patient from Greece. This strain harbored a new pI 9.1 plasmid-mediated AmpC β-lactamase with unusually high levels of hydrolytic activity for cefoxitin and cefotetan that we named MOX-2. Sequencing of
bla
MOX-2
revealed 93.2, 92.9, 92.7, and 73.1% identities with the deduced amino acid sequences of CMY-8, MOX-1, CMY-1, and the AmpC β-lactamase of
Aeromonas sobria
, respectively.
Fluorescence Assay for Studying the Ability of Macrolides To Induce Production of Ribosomal Methylase
Authors: Clarebout Gervais , Leclercq Roland
Published: July 2002

A screening assay to test the inducing capacity of macrolides by fusing the attenuator of the inducible
erm
(B) gene from
Streptococcus pneumoniae
HM28 with the
gfpmut1
gene has been designed. Fluorescence was detected under UV light around disks impregnated with inducer macrolides (erythromycin or azithromycin) but not with noninducer ketolides. Induction could be quantified by fluorometry.
Structure-Function Relationship Studies on the Frog Skin Antimicrobial Peptide Tigerinin 1: Design of Analogs with Improved Activity and Their Action on Clinical Bacterial Isolates
Authors: Sitaram Narasimhaiah , Sai Korrapati Purna , Singh Shashi , Sankaran Krishnan , Nagaraj Ramakrishnan
Published: July 2002

Structure-function relationships in antimicrobial peptides have been extensively investigated in order to obtain improved analogs. Most of these studies have targeted either α-helical peptides or β-sheet peptides with multiple disulfide bridges. Tigerinins are short, nonhelical antimicrobial peptides with a single disulfide bridge. In this study, we have synthesized several analogs of tigerinin 1 with an aim to understand the structural basis of activity as well as improve its activity. The studies demonstrate that the loop structure of tigerinin 1 is essential for its optimal activity. However, linearization with increased cationic charges can compensate for loss of loop structure to some extent. Morphology of the cells after treatment with the active analogs shows extensive leakage of cytoplasmic contents. Tigerinin 1 and two of its analogs exhibit impressive activity against a variety of clinical bacterial isolates.
GF120918, a P-Glycoprotein Modulator, Increases the Concentration of Unbound Amprenavir in the Central Nervous System in Rats
Authors: Edwards Jeffrey E. , Brouwer Kenneth R. , McNamara Patrick J.
Published: July 2002

The goal of this study was to determine the distribution of unbound amprenavir in the central nervous system (CNS) of rats. The concentration of unbound amprenavir in the extracellular fluid of the brain and the blood was examined in the presence and absence of the MDR modulator GF120918 by microdialysis. The brain-to-blood ratio of amprenavir in the absence and presence of GF120918 was found to be significantly different (
P
< 0.003; 0.076 and 0.617, respectively). The use of the MDR modulator GF120918 could potentially increase the penetration of human immunodeficiency virus protease inhibitors into the CNS.
Inhibition of Cyclin-Dependent Kinase 1 by Purines and Pyrrolo[2,3-d]Pyrimidines Does Not Correlate with Antiviral Activity
Authors: Evers David L. , Breitenbach Julie M. , Borysko Katherine Z. , Townsend Leroy B. , Drach John C.
Published: July 2002

We have previously shown that a series of nonnucleoside pyrrolo[2,3-d]pyrimidines selectively inhibit the replication of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). These compounds act at the immediate-early or early stage of HCMV replication and have antiviral properties somewhat similar to those of roscovitine and olomoucine, specific inhibitors of cyclin-dependent kinases (cdks). In the present study we examine the hypothesis that pyrrolo[2,3-d]pyrimidines exert their antiviral effects by inhibition of cellular cdks. Much higher concentrations of a panel of pyrrolo[2,3-d]pyrimidine nucleoside analogs with antiviral activity were required to inhibit recombinant cdk1/cyclin B compared to the submicromolar concentrations required to inhibit HCMV and HSV-1 replication. 4,6-Diamino-5-cyano-7-(2-phenylethyl)pyrrolo[2,3-d]pyrimidine (compound 1369) was the best inhibitor of cdk1 and cyclin B, with a 50% inhibitory concentration (IC50; 14 μM) similar to that of roscovitine; it was competitive with respect to ATP (Ki= 14 μM). The potency of compound 1369 against cdk1 and cyclin B was similar to its cytotoxicity (IC50s, 32 to 100 μM) but not its antiviral efficacy (IC50s, 0.02 to 0.3 μM). Thus, our results indicated the null hypothesis. In contrast, roscovitine was only weakly active against HSV-1 (IC50, 38 μM) and HCMV (IC50, 40 μM). These values were similar to those derived by cytotoxicity and cell growth inhibition assays, thereby suggesting that roscovitine is not a selective antiviral. Therefore, we propose that inhibition of cdk1 and cyclin B is not responsible for selective antiviral activity and that pyrrolo[2,3-d]pyrimidines constitute novel pharmacophores which compete with ATP to inhibit cdk1 and cyclin B.
Comparison of Visual and Spectrophotometric Methods of Broth Microdilution MIC End Point Determination and Evaluation of a Sterol Quantitation Method for In Vitro Susceptibility Testing of Fluconazole and Itraconazole against Trailing and Nontrailing
Candida
Isolates
Authors: Arthington-Skaggs Beth A. , Lee-Yang Wendy , Ciblak Meral A. , Frade Joao P. , Brandt Mary E. , Hajjeh Rana A. , Harrison Lee H. , Sofair Andre N. , Warnock and David W.
Published: July 2002

Visual determination of MIC end points for azole antifungal agents can be complicated by the trailing growth phenomenon. To determine the incidence of trailing growth, we performed testing of in vitro susceptibility to fluconazole and itraconazole using the National Committee for Clinical Laboratory Standards broth microdilution M27-A reference procedure and 944 bloodstream isolates of seven
Candida
spp., obtained through active population-based surveillance between 1998 and 2000. Of 429
C. albicans
isolates, 78 (18.2%) showed trailing growth at 48 h in tests with fluconazole, and 70 (16.3%) showed trailing in tests with itraconazole. Of 118
C. tropicalis
isolates, 70 (59.3%) showed trailing growth in tests with fluconazole, and 35 (29.7%) showed trailing in tests with itraconazole. Trailing growth was not observed with any of the other five
Candida
spp. tested (
C. dubliniensis
,
C. glabrata
,
C. krusei
,
C. lusitaniae
, and
C. parapsilosis
). To confirm whether or not isolates that showed trailing growth in fluconazole and/or itraconazole were resistant in vitro to these agents, all isolates that showed trailing growth were retested by the sterol quantitation method, which measures cellular ergosterol content rather than growth inhibition after exposure to azoles. By this method, none of the trailing isolates was resistant in vitro to fluconazole or itraconazole. For both agents, a 24-h visual end point or a spectrophotometric end point of 50% reduction in growth relative to the growth control after 24 or 48 h of incubation correlated most closely with the result of sterol quantitation. Our results indicate that MIC results determined by either of these end point rules may be more predictive of in vivo outcome for isolates that give unclear visual end points at 48 h due to trailing growth.
Small-Colony Mutants of
Staphylococcus aureus
Allow Selection of Gyrase-Mediated Resistance to Dual-Target Fluoroquinolones
Authors: Pan Xiao-Su , Hamlyn Penelope J. , Talens-Visconti Raquel , Alovero Fabiana L. , Manzo Ruben H. , Fisher L. Mark
Published: July 2002

Fluoroquinolones acting equally through DNA gyrase and topoisomerase IV in vivo are considered desirable in requiring two target mutations for emergence of resistant bacteria. To investigate this idea, we have studied the response of
Staphylococcus aureus
RN4220 to stepwise challenge with sparfloxacin, a known dual-target agent, and with NSFQ-105, a more potent sulfanilyl fluoroquinolone that behaves similarly. First-step mutants were obtained with both drugs but only at the MIC. These mutants exhibited distinctive small-colony phenotypes and two- to fourfold increases in MICs of NSFQ-105, sparfloxacin, and ciprofloxacin. No changes were detected in the quinolone resistance-determining regions of the
gyrA
,
gyrB
,
grlA
, or
grlB
gene. Quinolone-induced small-colony mutants shared the delayed coagulase response but not the requirement for menadione, hemin, or thymidine characteristic of small-colony variants, a subpopulation of
S. aureus
that is often defective in electron transport. Second-step mutants selected with NSFQ-105 had
gyrA
(S84L) alterations; those obtained with sparfloxacin carried a
gyrA
(D83A) mutation or a novel
gyrB
deletion (ΔRKSAL, residues 405 to 409) affecting a trypsin-sensitive region linking functional domains of
S. aureus
GyrB. Each mutation was associated with four- to eightfold increases in MICs of NSFQ-105 and sparfloxacin, but not of ciprofloxacin, which we confirm targets topoisomerase IV. The presence of wild-type
grlB
-
grlA
gene sequences in second-step mutants excluded involvement of topoisomerase IV in the small-colony phenotype. Growth revertants retaining mutant
gyrA
or
gyrB
alleles were quinolone susceptible, indicating that resistance to NSFQ-105 and sparfloxacin was contingent on the small-colony mutation. We propose that small-colony mutations unbalance target sensitivities, perhaps through altered ATP or topoisomerase levels, such that gyrase becomes the primary drug target. Breaking of target parity by genetic or physiological means eliminates the need for two target mutations and provides a novel mechanism for stepwise selection of quinolone resistance.
Novel Nucleoside Analogue MCC-478 (LY582563) Is Effective against Wild-Type or Lamivudine-Resistant Hepatitis B Virus
Authors: Ono-Nita Suzane Kioko , Kato Naoya , Shiratori Yasushi , Carrilho Flair José , Omata Masao
Published: July 2002

The emergence of resistant hepatitis B virus (HBV) with the L528M mutation and/or the M552V and M552I mutations in the polymerase gene following long-term lamivudine treatment is becoming an important clinical problem. The aim of this study was to investigate the susceptibility of wild-type and lamivudine-resistant HBV to MCC-478 (LY582563), a novel nucleoside analogue derivative of phosphonomethoxyethyl purine. The susceptibility of wild-type HBV and lamivudine-resistant mutants (M552I, M552V, and L528M/M552V) to MCC-478 was examined by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, and the effective concentration required to reduce replication by 50% (EC50) was determined. The replicative intermediates of wild-type and lamivudine-resistant mutants were progressively diminished by treatment with increasing doses of MCC-478. The MCC-478 EC50s were 0.027 μM for wild-type HBV (about 20 times more efficient than lamivudine), 2.6 μM for M552I, 3.3 μM for M552V, and 2.0 μM for L528M/M552V. Wild-type HBV and lamivudine-resistant mutants are susceptible to MCC-478. MCC-478 appears to be a candidate for the treatment of HBV infection and exhibits potent activity against lamivudine-resistant HBV.
In Vitro Activities of Quinupristin-Dalfopristin and Cefepime, Alone and in Combination with Various Antimicrobials, against Multidrug-Resistant Staphylococci and Enterococci in an In Vitro Pharmacodynamic Model
Authors: Allen George P. , Cha Raymond , Rybak Michael J.
Published: July 2002

Use of combinations of antimicrobials that together achieve synergistic activities against targeted microorganisms is one potential strategy for overcoming bacterial resistance. As the incidence of infections caused by multidrug-resistant staphylococci and enterococci increases, the importance of devising additional synergistic drug combinations for these bacteria is magnified. We evaluated a number of antimicrobial combinations, with a focus on quinupristin-dalfopristin (Q-D), cefepime, and linezolid, using a previously described in vitro pharmacodynamic model. The combination of Q-D with either linezolid or vancomycin, as well as the combination of cefepime-vancomycin, resulted in enhanced killing (≥2-log10increase in killing versus the most-active single agent) against methicillin-resistantStaphylococcus aureus(MRSA) 494. An improved effect (<2 log10kill increase in kill) against MRSA 494 was noted for cefepime plus either Q-D or linezolid, as well as linezolid-vancomycin. Similar relationships were observed for a methicillin-susceptibleS. aureusisolate (isolate 1199). Against methicillin-resistantS. epidermidisR444, enhanced killing was achieved with the combination of cefepime-linezolid, while improvement was noted for vancomycin with either cefepime or linezolid. The combination of cefepime and vancomycin also achieved enhanced killing against a glycopeptide-intermediate-susceptibleS. aureusisolate (isolate 992). The combination of linezolid and doxycycline achieved an enhanced effect against vancomycin-resistantEnterococcus faecalis(VREFc) andE. faecium. Q-D plus ampicillin or linezolid resulted in similar enhancement of activity against the VREFc isolate. The results of this study suggest a number of novel antimicrobial combinations that may be useful against staphylococci and enterococci. Combination regimens including cefepime, Q-D, and/or linezolid warrant further investigation for the treatment of refractive infections due to multidrug-resistant gram-positive pathogens.
Therapeutic Efficacies of GW471552 and GW471558, Two New Azasordarin Derivatives, against Pneumocystosis in Two Immunosuppressed-Rat Models
Authors: Jimenez Elena , Martínez Antonio , Aliouat El Moukhtar , Caballero Jesus , Dei-Cas Eduardo , Gargallo-Viola Domingo
Published: July 2002

Two new azasordarins, GW471552 and GW471558, were studied in vivo for treatment of
Pneumocystis carinii
pneumonia. In the Wistar rat spontaneous pneumonia model, both azasordarins significantly reduced the number of
P. carinii
cysts per gram of lung homogenate when administered at 1 mg/kg of body weight twice a day for 10 days. In a nude rat inoculation model, both compounds showed therapeutic efficacy at 0.25 mg/kg twice a day for 10 days.
Longitudinal Assessment of Antipneumococcal Susceptibility in the United States
Authors: Jones Mark E. , Karlowsky James A. , Blosser-Middleton Renée , Critchley Ian A. , Karginova Elena , Thornsberry Clyde , Sahm Daniel F.
Published: July 2002

The prevalence of antimicrobial resistance among 4,940 U.S. pneumococcal isolates collected during 1999 was as follows: penicillin, 16.2%; amoxicillin-clavulanate, 12.2%; cefuroxime, 28.1%; ceftriaxone, 3.6%; trimethoprim-sulfamethoxazole, 30.3%; azithromycin, 21.4%; levofloxacin, 0.6%; and moxifloxacin, 0.1%. Compared to the previous 1997-1998 study (Jones et al., Antimicrob. Agents Chemother.
44:
2645-2652, 2000), increases were noted for resistance to penicillin (3.7%;
P
< 0.001), amoxicillin-clavulanate (3.9%;
P
< 0.001), cefuroxime (5.7%;
P
< 0.001), azithromycin (2.4%;
P
= 0.014), trimethoprim-sulfamethoxazole (15.4%;
P
< 0.001), and levofloxacin (0.3%;
P
= 0.017). Resistance to ceftriaxone (0.1%;
P
= 0.809) and moxifloxacin (0.03%;
P
= 0.570) decreased. Concurrently, multidrug resistance increased (
P
< 0.001) from 6.3% to 11.3%.
Incidence, Epidemiology, and Characteristics of Quinolone- Nonsusceptible
Streptococcus pneumoniae
in Croatia
Authors: Pankuch Glenn A. , Bozdogan Bülent , Nagai Kensuke , Tambić-Andrašević Arjana , Schoenwald Slavko , Tambić Tera , Kalenić Smilja , Pleško Sanja , Tepeš Nastja K. , Kotarski Zdenka , Payerl-Pal Marina , Appelbaum Peter C.
Published: July 2002

Among 585
Streptococcus pneumoniae
strains isolated in 22 Croatian hospitals 21 strains (3.6%) were quinolone nonsusceptible. MICs of all quinolones were high for seven strains tested with the same serotype (23F) and mutations in
gyrA
,
parC
, and
parE
. The remaining 14 strains were more heterogeneous and had mutations only in
parC
and/or
parE
, and the MICs of quinolones were lower for these strains.
Biguanide-Atovaquone Synergy against
Plasmodium falciparum
In Vitro
Authors: Jones K. , Ward S. A.
Published: July 2002

The synergistic potential of a range of biguanides, their triazine metabolites, tetracyclines, and pyrimethamine in combination with atovaquone has been assessed. All five biguanides tested interacted synergistically with atovaquone against
Plasmodium falciparum
in vitro. All of the other compounds tested were either additive or antagonistic.
Noninvasive Quantitative Measurement of Bacterial Growth in Porous Media under Unsaturated-Flow Conditions
Authors: Yarwood R. R. , Rockhold M. L. , Niemet M. R. , Selker J. S. , Bottomley P. J.
Published: July 2002

Glucose-dependent growth of the
luxCDABE
reporter bacterium
Pseudomonas fluorescens
HK44 was monitored noninvasively in quartz sand under unsaturated-flow conditions within a 45- by 56- by 1-cm two-dimensional light transmission chamber. The spatial and temporal development of growth were mapped daily over 7 days by quantifying salicylate-induced bioluminescence. A nonlinear model relating the rate of increase in light emission after salicylate exposure to microbial density successfully predicted growth over 4 orders of magnitude (
r
2
= 0.95). Total model-predicted growth agreed with growth calculated from the mass balance of the system by using previously established growth parameters of HK44 (predicted, 1.2 × 10
12
cells; calculated, 1.7 × 10
12
cells). Colonization expanded in all directions from the inoculation region, including upward migration against the liquid flow. Both the daily rate of expansion of the colonized zone and the population density of the first day's growth in each newly colonized region remained relatively constant throughout the experiment. Nonetheless, substantial growth continued to occur on subsequent days in the older regions of the colonized zone. The proportion of daily potential growth that remained within the chamber declined progressively between days 2 and 7 (from 97 to 13%). A densely populated, anoxic region developed in the interior of the colonized zone even though the sand was unsaturated and fresh growth medium continued to flow through the colonized zone. These data illustrate the potential of a light transmission chamber, bioluminescent bacteria, and sensitive digital camera technology to noninvasively study real-time hydrology-microbiology interactions associated with unsaturated flow in porous media.
Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and
Haemophilus influenzae
Type b
Authors: Pickering Jerry W. , Martins Thomas B. , Schroder M. Carl , Hill Harry R.
Published: July 2002

We developed a multiplexed indirect immunofluorescence assay for antibodies to
Haemophilus influenza
type b (Hib) polysaccharide and the toxoids of
Clostridium tetani
(Tet) and
Corynebacterium diphtheriae
(Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (≥0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (≥0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (≥1.0 μg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (
R
2
) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.
Widal Test in Diagnosis of Typhoid Fever in Turkey
Authors: Willke Ayse , Ergonul Onder , Bayar Banu
Published: July 2002

We studied the value of the Widal tube agglutination test for the diagnosis of typhoid fever. The subjects were all adults >18 years of age and were divided into four groups: (i) 317 healthy blood donor controls, (ii) 31 bacteriologically confirmed patients with
Salmonella enterica
serotype Typhi, (iii) 21 patients with a clinical diagnosis of typhoid fever, and (iv) 41 febrile nontyphoid patients. Blood donor controls were screened with a slide agglutination test for the
Salmonella enterica
serotype Typhi O and H antigens, and positives were then tested with the Widal test. Acute- and convalescent-phase sera from patients in groups 2, 3, and 4 were obtained 7 to 10 days apart and tested by the Widal test. Using a cutoff of ≥1/200 for the O antigen test performed on acute-phase serum gave a sensitivity of 52% and a specificity of 88% with a positive predictive value (PPV) of 76% and a negative predictive value (NPV) of 71%. This increased to 90% sensitivity and specificity with a PPV of 88% and an NPV of 93% when the convalescent-phase serum was tested. We concluded that O and H agglutinin titers of ≥1/200 are of diagnostic significance. The Widal test is easy, inexpensive, and relatively noninvasive. It can be of diagnostic value when blood cultures are not available or practical. The results must be interpreted cautiously because of the low sensitivity of the test. The Widal test done on convalescent-phase serum gave more-reliable results with higher specificity and sensitivity.
In Vitro Cultivation of
Cryptosporidium
Species
Authors: Arrowood Michael J.
Published: July 2002

The in vitro cultivation of protozoan parasites of the genus
Cryptosporidium
has advanced significantly in recent years. These obligate, intracellular parasites colonize the epithelium of the digestive and respiratory tracts, are often difficult to obtain in significant numbers, produce durable oocysts that defy conventional chemical disinfection methods, and are persistently infectious when stored at refrigerated temperatures (4 to 8°C). While continuous culture and efficient life cycle completion (oocyst production) have not yet been achieved in vitro, routine methods for parasite preparation and cell culture infection and assays for parasite life cycle development have been established. Parasite yields may be limited, but in vitro growth is sufficient to support a variety of research studies, including assessing potential drug therapies, evaluating oocyst disinfection methods, and characterizing life cycle stage development and differentiation.
Human Parvovirus B19
Authors: Heegaard Erik D. , Brown Kevin E.
Published: July 2002

Parvovirus B19(B19) was discovered in 1974 and is the only member of the familyParvoviridaeknown to be pathogenic in humans. Despite the inability to propagate the virus in cell cultures, much has been learned about the pathophysiology of this virus, including the identification of the cellular receptor (P antigen), and the control of the virus by the immune system. B19 is widespread, and manifestations of infection vary with the immunologic and hematologic status of the host. In healthy immunocompetent individuals B19 is the cause of erythema infectiosum and, particularly in adults, acute symmetric polyarthropathy. Due to the tropism of B19 to erythroid progenitor cells, infection in individuals with an underlying hemolytic disorder causes transient aplastic crisis. In the immunocompromised host persistent B19 infection is manifested as pure red cell aplasia and chronic anemia. Likewise, the immature immune response of the fetus may render it susceptible to infection, leading to fetal death in utero, hydrops fetalis, or development of congenital anemia. B19 has also been suggested as the causative agent in a variety of clinical syndromes, but given the common nature, causality is often difficult to infer. Diagnosis is primarily based on detection of specific antibodies by enzyme-linked immunosorbent assay or detection of viral DNA by dot blot hybridization or PCR. Treatment of persistent infection with immunoglobulin reduces the viral load and results in a marked resolution of anemia. Vaccine phase I trials show promising results.
Recurrent Variable Region Gene Usage and Somatic Mutation in the Human Antibody Response to the Capsular Polysaccharide ofStreptococcus pneumoniaeType 23F
Authors: Zhou Jianhui , Lottenbach Kathleen R. , Barenkamp Stephen J. , Lucas Alexander H. , Reason Donald C.
Published: July 2002

Combinatorial cloning and expression library analysis were used to isolate human antibody Fab fragments specific for the capsular polysaccharide ofStreptococcus pneumoniaeserotype 23F. Thirty 23F-specific Fabs were isolated from seven vaccinated donors, and the sequences of the heavy (H)- and light (L)-chain variable regions were determined. All individuals utilized either the Vκ A23 L chain, the Vκ L6 L chain, or both chains in forming the 23F-specific combining site. Vκ A23 L chains paired primarily with VH3-23 H chains. Vκ L6 L chains were more promiscuous in heavy-chain usage between individuals. Both H and L chains were mutated, primarily in the complementarity-determining regions, compared to their closest germ line counterpart, suggesting a recall response that has undergone affinity maturation. H-chain isotypes were reflective of those found in the serum. Shared somatic modifications demonstrated that immunoglobulin G2 (IgG2) and IgA antibodies arose from the same somatically matured B cell. Our results indicate that the response to the serotype 23F pneumococcal capsular polysaccharide is oligoclonal within the individual, with one or two paratope families accounting for the majority of expressed antibody. We also determined that, in spite of the combinatorial diversity available to the immune system, the 23F-specific response is highly restricted at the population level, with the same two L-chain-determined paratope families recurring in all individuals. Lastly, analysis of the isolated Fabs indicate all have undergone extensive somatic mutation, as well as class switch, maturational events that presumably require the participation of T cells.
Disruption of the Gene Homologous to MammalianNramp1inMycobacterium tuberculosisDoes Not Affect Virulence in Mice
Authors: Boechat Neio , Lagier-Roger Béatrice , Petit Stéphanie , Bordat Yann , Rauzier Jean , Hance Allan J. , Gicquel Brigitte , Reyrat Jean-Marc
Published: July 2002

Natural-resistance-associated macrophage protein 1 (Nramp1) is a divalent cation transporter belonging to a family of transporter proteins highly conserved in eukaryotes and prokaryotes. Mammalian and bacterial transporters may compete for essential metal ions during mycobacterial infections. The mycobacterialNramphomolog may therefore be involved inMycobacterium tuberculosisvirulence. Here, we investigated this possibility by inactivating theM. tuberculosis Nramp1gene (Mramp) by allelic exchange mutagenesis. Disruption ofMrampdid not affect the extracellular growth of bacteria under standard conditions. However, theMrampmutation was associated with growth impairment under conditions of limited iron availability. TheMrampmutant displayed no impairment of growth or survival in macrophages derived from mouse bone marrow or inNramp1+/+andNramp1−/−congenic murine macrophage cell lines. Following intravenous challenge in BALB/c mice, counts of parental andMrampmutant strains were similar in the lungs and spleens of the animals at all time points studied. These results indicate thatMrampdoes not contribute to the virulence ofM. tuberculosisin mice.
Identification of Chromosomal
Shigella flexneri
Genes Induced by the Eukaryotic Intracellular Environment
Authors: Runyen-Janecky L. J. , Payne S. M.
Published: July 2002

Upon entry into the eukaryotic cytosol, the facultative intracellular bacterium
Shigella flexneri
is exposed to an environment that may necessitate the expression of particular genes for it to survive and grow intracellularly. To identify genes that are induced in response to the intracellular environment, we screened a library containing fragments of the
S. flexneri
chromosome fused to a promoterless green fluorescent protein gene (
gfp
). Bacteria containing promoter fusions that had a higher level of
gfp
expression when
S. flexneri
was intracellular (in Henle cells) than when
S. flexneri
was extracellular (in Luria-Bertani broth) were isolated by using fluorescence-activated cell sorting. Nine different genes with increased expression in Henle cells were identified. Several genes (
uhpT
,
bioA
, and
lysA
) were involved in metabolic processes. The
uhpT
gene, which encoded a sugar phosphate transporter, was the most frequently isolated gene and was induced by glucose-6-phosphate in vitro. Two of the intracellularly induced genes (
pstS
and
phoA
) encode proteins involved in phosphate acquisition and were induced by phosphate limitation in vitro. Additionally, three iron-regulated genes (
sufA
,
sitA
, and
fhuA
) were identified. The
sufA
promoter was derepressed in iron-limiting media and was also induced by oxidative stress. To determine whether intracellularly induced genes are required for survival or growth in the intracellular environment, we constructed mutations in the
S. flexneri uhpT
and
pstS
genes by allelic exchange. The
uhpT
mutant could not use glucose-6-phosphate as a sole carbon source in vitro but exhibited normal plaque formation on Henle cell monolayers. The
pstS
mutant had no apparent growth defect in low-phosphate media in vitro but formed smaller plaques on Henle cell monolayers than the parent strain. Both mutants were as effective as the parent strain in inducing apoptosis in a macrophage cell line.
ADP and Other Metabolites Released fromAcanthamoeba castellaniiLead to Human Monocytic Cell Death through Apoptosis and Stimulate the Secretion of Proinflammatory Cytokines
Authors: Mattana A. , Cappai V. , Alberti L. , Serra C. , Fiori P. L. , Cappuccinelli P.
Published: July 2002

Monocytes/macrophages are thought to be involved inAcanthamoebainfections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released byAcanthamoeba castellaniitrophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium byA. castellanii, interacting with specific P2y2purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca2+]i, morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-α). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-α release induced by amoebic ADP were blocked by the P2y2inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1β. These findings suggest that pathogenic free-livingA. castellaniiby release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.
Immunochemical Properties of the Staphylococcal Poly-
N
-Acetylglucosamine Surface Polysaccharide
Authors: Maira-Litrán Tomás , Kropec Andrea , Abeygunawardana C. , Joyce Joseph , Mark George , Goldmann Donald A. , Pier Gerald B.
Published: July 2002

Staphylococcus aureus
and
Staphylococcus epidermidis
often elaborate adherent biofilms, which contain the capsular polysaccharide-adhesin (PS/A) that mediates the initial cell adherence to biomaterials. Biofilm cells produce another antigen, termed polysaccharide intercellular adhesin (PIA), which is composed of a ∼28 kDa soluble linear β(1-6)-linked
N
-acetylglucosamine. We developed a new method to purify PS/A from
S. aureus
MN8m, a strain hyperproducing PS/A. Using multiple analytical techniques, we determined that the chemical structure of PS/A is also β(1-6)-
N
-acetylglucosamine (PNAG). We were unable to find
N
-succinylglucosamine residues in any of our preparations in contrast to previously reported findings (D. McKenney, K. Pouliot, Y. Wang, V. Murthy, M. Ulrich, G. Doring, J. C. Lee, D. A Goldmann, and G. B. Pier, Science 284:1523-1527, 1999). PNAG was produced with a wide range of molecular masses that could be divided into three major fractions with average molecular masses of 460 kDa (PNAG-I), 100 kDa (PNAG-II), and 21 kDa (PNAG-III). The purified antigens were not soluble at neutral pH unless first dissolved in 5 M HCl and then neutralized with 5 M NaOH. PNAG-I was very immunogenic in rabbits, but the responses of individual animals were variable. Immunization of mice with various doses (100, 50, or 10 μg) of PNAG-I, -II, and -III demonstrated that only PNAG-I was able to elicit an immunoglobulin G (IgG) immune response with the highest titers obtained with 100-μg dose. When we purified a small fraction of PNAG with a molecular mass of ∼780 kDa (PNAG-780) from PNAG-I, significantly higher IgG titers than those in mice immunized with the same doses of PNAG-I were obtained, suggesting the importance of the molecular mass of PNAG in the antibody response. These results further clarify the chemical structure of PS/A and help to differentiate it from PIA on the basis of immunogenicity, molecular size, and solubility.
High-Level Expression of the Malaria Blood-Stage Vaccine CandidatePlasmodium falciparumApical Membrane Antigen 1 and Induction of Antibodies That Inhibit Erythrocyte Invasion
Authors: Kocken Clemens H. M. , Withers-Martinez Chrislaine , Dubbeld Martin A. , van der Wel Annemarie , Hackett Fiona , Blackman Michael J. , Thomas Alan W.
Published: July 2002

Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression ofPlasmodium falciparumFVO strain AMA-1 (PfAMA-1) inPichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites inPlasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologousP. falciparumby immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologousP. falciparum.
Sequential Expression of the Neuropeptides Substance P and Somatostatin in Granulomas Associated with Murine Cysticercosis
Authors: Robinson Prema , White A. Clinton , Lewis Dorothy E. , Thornby John , David Elliott , Weinstock Joel
Published: July 2002

Neurocysticercosis, a parasitic infection of the human central nervous system caused byTaenia solium, is a leading cause of seizures. Seizures associated with neurocysticercosis are caused mainly by the host inflammatory responses to dying parasites in the brain parenchyma. We previously demonstrated sequential expression of Th1 cytokines in early-stage granulomas, followed by expression of Th2 cytokines in later-stage granulomas in murine cysticercosis. However, the mechanism leading to this shift in cytokine response in the granulomas is unknown. Neuropeptides modulate cytokine responses and granuloma formation in murine schistosomiasis. Substance P (SP) induces Th1 cytokine expression and granuloma formation, whereas somatostatin inhibits the granulomatous response. We hypothesized that neuropeptides might play a role in regulation of the granulomatous response in cysticercosis. To test this hypothesis, we compared expression of SP and expression of somatostatin in murine cysticercal granulomas by using in situ hybridization and immunohistochemistry. We also compared expression with granuloma stage. Expression of SP mRNA was more frequent in the early-stage granulomas than in the late-stage granulomas (34 of 35 early-stage granulomas versus 1 of 13 late-stage granulomas). By contrast, somatostatin was expressed primarily in later-stage granulomas (13 of 14 late-stage granulomas versus 2 of 35 early-stage granulomas). The median light microscope grade of SP mRNA expression in the early-stage granulomas was significantly higher than that in the late-stage granulomas (P= 0.008, as determined by the Wilcoxon signed rank test). By contrast, somatostatin mRNA expression was higher at later stages (P= 0.008, as determined by the Wilcoxon signed rank test). SP and somatostatin are therefore temporally expressed in granulomas associated with murine cysticercosis, which may be related to differential expression of Th1 and Th2 cytokines.
Identification ofEscherichia coliOuter Membrane Protein A Receptor on Human Brain Microvascular Endothelial Cells
Authors: Prasadarao Nemani V.
Published: July 2002

NeonatalEscherichia colimeningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible forEscherichia colitraversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) forE. coliinvasion. Ecgp was partially purified by wheat germ agglutinin andMaackia amurensislectin (MAL) affinity chromatography. The MAL affinity-purified HBMEC proteins bound to OmpA+E. colibut not to OmpA−E. coli. In addition, the deglycosylated MAL-bound proteins still interact with OmpA+E. coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC. Interestingly, the MAL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA+E. coliin addition to Ecgp. Further, the 65-kDa protein was shown to be a cleavage product of Ecgp. Immunocytochemistry of HBMEC infected with OmpA+E. coliby using anti-Ecgp antibody suggests that Ecgp clusters at theE. colientry site. Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp inE. colimeningitis. Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface. In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier. These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC.
Effects of Oral Vaccination and Immunomodulation by Cholera Toxin on ExperimentalHelicobacter pyloriInfection, Reinfection, and Gastritis
Authors: Raghavan S. , Svennerholm A.-M. , Holmgren J.
Published: July 2002

Therapeutic vaccination is an attractive strategy to control infection and disease caused byHelicobacter pylori. In mice infected withH. pyloriwe have studied the protective effect of oral immunization with anH. pylorilysate preparation given together with the mucosal adjuvant cholera toxin (CT), both against the initial infection and against a later reinfection challenge. We have also examined the effects of treatment with the CT adjuvant alone onH. pyloriinfection and reinfection. Specific immunization with lysate was found to result in a sixfold reduction of the extent (bacterial load) of the primary infection and also to provide similar levels of protection against reinfection. However, these effects were associated with severe postimmunization gastritis. In contrast, oral treatment with CT alone at the time of initial infection, while unable to suppress the initial infection, gave rise to a 20-fold reduction in bacterial load upon reinfection without causing any associated gastric inflammation. Both the infected animals that were specifically immunized and those that were treated with CT only displayed increased in vitro proliferative responses of mononuclear cells toH. pyloriantigens. Antibody levels in response toH. pyloriwere on the other hand only marginally increased after treatment with CT, whereas they were markedly elevated after immunization with lysate plus CT, with a rise in both (Th2-driven) immunoglobulin G1 (IgG1) and, especially, (Th1-driven) IgG2a antibodies. The results illustrate the complex balance between protection and harmful inflammation after postinfection vaccination againstH. pylorias studied in a mouse model.
Old Mice Express a Transient Early Resistance to Pulmonary Tuberculosis That Is Mediated by CD8 T Cells
Authors: Turner Joanne , Frank Anthony A. , Orme Ian M.
Published: July 2002

During the natural aging process the immune system undergoes many alterations. In particular, both the CD4 and CD8 T-cell compartments become compromised, and these changes have serious implications for the capacity of the elderly to control infection. As a result, the elderly are more susceptible to many infectious diseases, including primary infection and reactivation of latent infections. In this study we addressed the capacity of old mice to control an infection withMycobacterium tuberculosisand to characterize the mechanism by which old mice, paradoxically, can express a transient early resistance to infection. This resistance was shown to be associated with the presence of CD8 T cells within the lungs that were capable of secreting gamma interferon, as illustrated by the demonstration that early resistance was lost in aged CD8 gene-disrupted mice. These studies therefore show that, despite a documented decline in general CD8 T-cell responsiveness in the elderly, a subset of CD8 T cells is an important early mediator of protection in the lungs of old mice that have been infected withM. tuberculosis.
Mobilization of Protein Kinase C in Macrophages Induced byListeria monocytogenesAffects Its Internalization and Escape from the Phagosome
Authors: Wadsworth Sandra J. , Goldfine Howard
Published: July 2002

Listeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC) are known virulence factors ofListeria monocytogenesin both tissue cultures and the murine model of infection. LLO is a member of a family of pore-forming cholesterol-dependent cytotoxins and is known to play an essential role in escape from the primary phagocytic vacuole of macrophages. PI-PLC plays an accessory role, in that PI-PLC mutants are partially defective in escape. We have shown that both of these molecules are essential for initiating rapid increases in the calcium level in the J774 murine macrophage cell line (S. J. Wadsworth and H. Goldfine, Infect. Immun.67:1770-1778, 1999). Here we show that both LLO and PI-PLC are required for translocation of protein kinase C δ (PKC δ) to the periphery of J774 cells and for translocation of PKC β II to early endosomes beginning within the first minute after addition of bacteria to the culture medium. Treatment with the calcium channel blocker SK&F 96365 inhibited translocation of PKC β II but not PKC δ. Our findings lead us to propose a host signaling pathway requiring LLO and the formation of diacylglycerol by PI-PLC in which calcium-independent PKC δ is responsible for the initial calcium signal and the subsequent PKC β II translocation. LLO-dependent translocation of PKC β I to early endosomes also occurs between 1 and 4 min after infection, but this occurs in the absence of PI-PLC. All of these signals were observed in cells that had not internalized bacteria. Blocking PKC β translocation with hispidin resulted in more rapid uptake of wild-type bacteria and greatly reduced escape from the primary phagocytic vacuoles of J774 cells.