bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamine Synthetase GlnA1 Is Essential for Growth of
Mycobacterium tuberculosis
in Human THP-1 Macrophages and Guinea Pigs
Authors: Tullius Michael V. , Harth Günter , Horwitz Marcus A.
Published: June 2003

To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of
Mycobacterium tuberculosis
, we constructed a
glnA1
mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for
l
-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of
l
-glutamine, the effective concentration of
l
-glutamine in the
M
.
tuberculosis
phagosome of THP-1 cells was ∼10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the
M
.
tuberculosis
phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the
M
.
tuberculosis glnA1
gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the
Salmonella enterica
serovar Typhimurium
glnA
gene, the mutant had only 1% of the GS activity of the
M
.
tuberculosis
wild-type strain because of poor expression of the
S
.
enterica
serovar Typhimurium GS in the heterologous
M
.
tuberculosis
host. Nevertheless, the strain complemented with
S
.
enterica
serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that
glnA1
is essential for
M
.
tuberculosis
virulence.
Deletion of a
Vibrio cholerae
ClC Channel Results in Acid Sensitivity and Enhanced Intestinal Colonization
Authors: Ding Yanpeng , Waldor Matthew K.
Published: June 2003

ClC chloride channels are found in all three kingdoms of life though little is known about their functions in prokaryotes. Here we investigated the role of a
Vibrio cholerae
ClC channel in acid resistance and intestinal colonization. The putative
V. cholerae
ClC channel was found to confer mild resistance to acid when pH was adjusted with HCl, but not with other acids. Surprisingly, a ClC channel deletion mutant exhibited enhanced intestinal colonization in infant mice.
Mutation of
sarA
in
Staphylococcus aureus
Limits Biofilm Formation
Authors: Beenken Karen E. , Blevins Jon S. , Smeltzer Mark S.
Published: June 2003

Mutation of
sarA
resulted in a reduced capacity to form a biofilm in six of the eight
Staphylococcus aureus
strains we tested (UAMS-1, UAMS-601, SA113, SC-01, S6C, and DB). The exceptions were Newman, which formed a poor biofilm under all conditions, and RN6390, which consistently formed a biofilm only after mutation of
agr
. Mutation of
agr
in other strains had little impact on biofilm formation. In every strain other than Newman, including RN6390, simultaneous mutation of
sarA
and
agr
resulted in a phenotype like that observed with the
sarA
mutants. Complementation studies using a
sarA
clone confirmed that the defect in biofilm formation was due to the
sarA
mutation.
Theory and Practice: A Conversation With Marilyn E. Parker
Authors: Fawcett Jacqueline
Published: June 2003

This edited transcript of an electronic mail dialogue with Marilyn E. Parker presents Dr. Parker’s ideas about nursing theory, nursing practice, and the relation between theory and practice.
Interruptibility of long call production in tamarins: implications for vocal control
Authors: Miller Cory T. , Flusberg Stephen , Hauser Marc D.
Published: June 2003

Vocal production can be highly deterministic, such that once the central nervous system generates a signal to call, the vocalization is emitted immune to external events. Conversely, vocal production can be modulated by auditory feedback such that interference or disruption can cause an individual to stop calling or, if it continues to call, for the acoustic morphology of the signal to change. To explore which of these models best accounts for the control of vocal production in non-human primates, we adapted an interruption technique originally developed for songbirds for use with a New World monkey species,the cotton-top tamarin (Saguinus oedipus). Results from a pilot experiment indicated that an auditory stimulus (white noise) was more effective than a visual stimulus (strobe light) at interrupting the tamarin's species-typical `combination long call (CLC)'. Data from a second experiment showed that although the duration of the auditory stimulus did not affect the proportion of interruptions that occurred, a 1000 ms white noise stimulus perturbed the temporal structure of the CLC to a greater extent than did a 250 ms white noise stimulus. Furthermore, when call production was interrupted,tamarins stopped vocalizing after the completion of a syllable, suggesting that the syllable represents a unit of organization within the call. Overall,these results provide evidence that tamarins can modify their vocal output based on external events, but the degree of vocal control is significantly less than in oscine songbirds.
Salvage of Failed Sauvé–Kapandji Procedure with an Ulnar Head Prosthesis: Report of Three Cases
Authors: DE SMET L. , PEETERS T.
Published: June 2003

Three failed Sauvé–Kapandji procedures were salvaged using an ulnar head prosthesis. At 7–22 month follow-up, all three patients were much improved. However, the stem of one implant subsequently fractured in a fall and this implant had to be removed.
The Classification of Phocomelia
Authors: TYTHERLEIGH-STRONG G. , HOOPER G.
Published: June 2003

We studied 24 patients with 44 phocomelic upper limbs. Only 11 limbs could be grouped in the classification system of Frantz and O’ Rahilly. The non-classifiable limbs were further studied and their characteristics identified. It is confirmed that phocomelia is not an intercalary defect.
Evaluation of Carpal Tunnel Release Using the Knifelight® Instrument
Authors: HELM R. H. , VAZIRI S.
Published: June 2003

We have performed a prospective randomized controlled trial to compare the results of open carpal tunnel release with those of carpal tunnel release using a Knifelight® (Stryker, Kalamazoo, MI). This is a new knife with its own battery-powered light source which enables the operation to be performed through a small incision in the palm of the hand. There were 43 patients in the open operation group and 39 in the Knifelight group. We found no difference in discomfort reported during surgery, in the operative time, in the grip strength measured at 2 and 6 weeks postoperatively or in the proportion of patients cured of their pre-operative symptoms. Patients in the Knifelight group had a statistically significant improvement in the time to return to work and in scar tenderness at 6 weeks post-operatively.
A New Clinical Severity Scoring System for Reflex Sympathetic Dystrophy of the Upper Limb
Authors: ZYLUK A.
Published: June 2003

A scoring system for the clinical severity of reflex sympathetic dystrophy (RSD) is proposed. The following signs and symptoms were considered to be significant: pain, reduction of finger flexion, swelling, temperature changes, discolouration, sensory disturbances, shoulder pain and loss of movement, increased sweating, and hair/nail growth changes. One point was assigned for strong expression of most of these features, half a point for moderate expression and no point if the feature was absent. A total score of 4 points was assumed, empirically, to indicate a mild degree of RSD, and the maximum score of 10 to indicate the most severe form of RSD. One hundred and forty-six patients with RSD of the hand were classified according to this system. Seventy-four patients (51%) had a score of 4–6, 51 patients (35%) a score of 6.5–8, and 21 patients (14%) a score of 8.5–10. Use of this system will allow more precise analysis of RSD patients and facilitate comparisons between studies.
The Position of the Centre of the Distal Radial Articular Surface in the Sagittal Plane: A Radiological Study
Authors: KRISHNAN K. M. , BASSI R. S. , DESHMUKH S. C.
Published: June 2003

Using a digital vernier calliper, measurements of the position of the centre of the distal radial articular surface in the sagittal plane with respect to the long axis of the distal radius were made on 50 lateral radiographs of normal wrists. In all 50 cases, the centre of the distal radial articular surface was palmar on the long axis of the radius. The mean value for this palmar position was 5.3 mm (44% of the radial shaft width). There was no correlation between the position of the centre of the distal radial articular surface and either the width of the radial shaft, the length of the articular surface of the distal radius or the age or sex of the individuals. However, the position of the centre of rotation was found to correlate with palmar tilt. The palmar position of the centre of the distal radial articular surface on the long axis of the radius may be biomechanically important.
Compression and Stretching of the Brachial Plexus in Thoracic Outlet Syndrome: Correlation Between Neuroradiographic Findings and Symptoms and Signs Produced by Provocation Manoeuvres
Authors: IDE J. , KATAOKA Y. , YAMAGA M. , KITAMURA T. , TAKAGI K.
Published: June 2003

In order to investigate the mechanism of nerve irritation in thoracic outlet syndrome (TOS), we studied 150 patients who presented with symptoms of neurologic TOS between 1985 and 1999. They first performed various provocative physical manoeuvres and then underwent injection of contrast medium into the supraclavicular part of the brachial plexus. Several of the provocative manoeuvres were then repeated and radiographs were again obtained. Based on the neuroradiographs, we identified three subsets of patients; those with only compression (type 1 TOS, n=27, 18%), those with combined compression and stretching (type 2 TOS, n=111, 74%), and those with only stretching (type 3 TOS, n=12, 8%). We were able to correlate the neuroradiological subsets with symptoms elicited by pre-radiographic provocative manoeuvres; in 92 patients (61%) these were elicited by traction manoeuvres. We conclude that stretching is an important factor of nerve irritation in TOS.
Palmar Plate Fixation of AO Type C2 Fracture of Distal Radius Using a Locking Compression Plate –A Biomechanical Study in a Cadaveric Model
Authors: LEUNG F. , ZHU L. , HO H. , LU W. W. , CHOW S. P.
Published: June 2003

The stability of palmar plate fixation using a locking compression T-plate was compared with that of a conventional palmar T-plate and a dorsal T-plate in a cadaveric model of an AO type C2 fracture of distal radius. The wrist axial load transmission through the radius was tested for each fixation. The results show that, under 100N axial load, the palmar locking compression T-plate restores stability comparable to that of the intact radius, and is superior to conventional palmar or dorsal T-plates.
Reconstruction of the Interosseous Ligament of the Forearm Reduces Load on the Radial Head in Cadavers
Authors: TOMAINO MATTHEW M. , PFAEFFLE JAMIE , STABILE KATHRYNE , LI ZONG-MING
Published: June 2003

Excision of the radial head after fracture may be complicated by longitudinal radio-ulnar instability (Essex-Lopresti lesion) if the forearm interosseous ligament has also been torn. In such cases proximal migration of the radius occurs, and ulnar impaction at the wrist and radiocapitellar contact at the elbow may impair function. Although metal radial head arthroplasties are now used for irreparable radial head fractures, the long-term clinical outcome may still be unsatisfactory because of excessive radiocapitellar load causing pain. Interosseous ligament reconstruction might improve outcome by restoring normal load transfer from the radius to ulna, but the biomechanical effect of reconstruction has not been reported. This study evaluated forearm load transfer following interosseous ligament reconstruction with an Achilles tendon allograft in a cadaveric model with the radial head intact. Interosseous ligament reconstruction reduced proximal radius loading by transferring force to the proximal ulna, but force transfer by the reconstruction was only half that by the intact ligament.
Dynamic Splinting After Extensor Tendon Repair in Zones V to VII
Authors: BRÜNER S. , WITTEMANN M. , JESTER A. , BLUMENTHAL K. , GERMANN G.
Published: June 2003

This retrospective study evaluates a dynamic active motion protocol for extensor tendon repairs in zones V to VII. Fifty-eight patients with 87 extensor tendon injuries were examined. Using Geldmacher’s and Kleinert and Verdan’s evaluation systems, the results were graded as “excellent” and “good” in more than 94%, and as “satisfactory” in the remainder. The need for secondary tenolysis was low (6%), and no other surgical complication occurred.
A Study of the Relative Responsiveness of Five Sensibility Tests for Assessment of Recovery after Median Nerve Injury and Repair
Authors: JEROSCH-HEROLD CHRISTINA
Published: June 2003

A longitudinal dynamic cohort study was conducted on patients with median nerve injuries to evaluate the relative responsiveness of five sensibility tests: touch threshold using the WEST (monofilaments), static two-point discrimination, locognosia, a pick-up test and an object recognition test. Repeated assessments were performed starting at 6 months after surgery. In order to compare the relative responsiveness of each test, effect size and the standard response mean were calculated for sensibility changes occurring between 6 and 18 months after repair.

Large effect sizes (>0.8) and standard response means (>0.8) were obtained for the WEST, locognosia, pick-up and object recognition tests. Two-point discrimination was hardly measurable at any time point and exhibited strong flooring effects. Further analysis of all time points was undertaken to assess the strength of the monotonic relationship between test scores and time elapsed since surgery. Comparison of monotonicity between the five tests indicated that the WEST performed best, whereas two-point discrimination performed worst. These results suggest that the monofilament test (WEST), locognosia test, Moberg pick-up test and tactile gnosis test capture sensibility changes over time well and should be considered for inclusion in the outcome assessment of patients with median nerve injury.
Nitric Oxide Production and Mononuclear Cell Nitric Oxide Synthase Activity in Malaria-Tolerant Papuan Adults
Authors: Boutlis Craig S. , Tjitra Emiliana , Maniboey Helena , Misukonis Mary A. , Saunders Jocelyn R. , Suprianto Sri , Weinberg J. Brice , Anstey Nicholas M.
Published: June 2003

Individuals living in regions of intense malaria transmission exhibit natural immunity that allows them to be without fever and other symptoms for most of the time despite frequent parasitization. Although this tolerance of parasitemia appears to be more effective in children than in adults (as evidenced by lower parasitemia fever thresholds with age), adults do exhibit a degree of tolerance but the mechanism(s) underlying this are unclear. Asymptomatic malaria-exposed children have higher levels of nitric oxide (NO) than children with severe disease, and NO has been proposed as a mediator of malarial tolerance. However, the ability of highly malaria-exposed asymptomatic adults to generate high-level basal NO is unknown, as is the relationship between NO and malaria tolerance in adults. The relationship between NO and malaria parasitemia was therefore determined in asymptomatic adults from Papua, Indonesia. Adults withPlasmodium falciparumparasitemia had markedly increased basal systemic NO production relative to aparasitemic Papuan controls, who in turn produced more NO than healthy controls from a region without malaria. Immunoglobulin E levels were universally elevated in malaria-exposed Papuan subjects, suggesting that the prevalence of intestinal parasitosis may be high and that nonmalarial infection may also contribute to high basal NO production. Basal peripheral blood mononuclear cell (PBMC) NO synthase activity was elevated in Papuans but poorly correlated with systemic NO production, suggesting that NO production in this setting arises not only from PBMCs but also from other tissue and cellular sources. NO production was associated with and may contribute to malaria tolerance in Papuan adults.
Overexpression of Interleukin-4 in Lungs of Mice Impairs Elimination ofHistoplasma capsulatum
Authors: Gildea Lucy A. , Gibbons Reta , Finkelman Fred D. , Deepe George S.
Published: June 2003

Protection against the pathogenic fungusHistoplasma capsulatumrequires Th1 cytokines. Since interleukin-4 (IL-4) can inhibit both Th1 cytokine production and activity, we examined the effects of overproduction of IL-4 in the lung on the course of pulmonary histoplasmosis. IL-4 lung transgenic mice manifested a higher fungal burden in their lungs, but not spleens, compared to wild-type infected controls. Despite the higher burden, the transgenic animals were ultimately capable of controlling infection. The adverse effects of IL-4 onH. capsulatumelimination were not observed during the early phase of infection (days 1 to 3) but were maximal at day 7 postinfection, prior to the induction of cell-mediated immunity. Analysis of total body and lung cytokine levels revealed that gamma interferon and tumor necrosis factor alpha production were not inhibited in the presence of excess IL-4. Our results with transgenic mice were supported by additional in vivo studies in which allergen induction of pulmonary IL-4 was associated with delayed clearance ofH. capsulatumyeast and increased fungal burden. These findings demonstrate that excess production of endogenous IL-4 modulates protective immunity toH. capsulatumby delaying clearance of the organism but does not prevent the generation of a Th1 response that ultimately controls infection.
The Largest Open Reading Frame (
pks12
) in the
Mycobacterium tuberculosis
Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis
Authors: Sirakova Tatiana D. , Dubey Vinod S. , Kim Hwa-Jung , Cynamon Michael H. , Kolattukudy Pappachan E.
Published: June 2003

The cell wall lipids in
Mycobacterium tuberculosis
are probably involved in pathogenesis. The largest open reading frame in the genome of
M. tuberculosis
H37Rv,
pks12
, is unique in that it encodes two sets of domains needed to produce fatty acids. A
pks12
-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the
pks12-
encoded sequence. Biochemical analysis using [1-
14
C]propionate as the radiotracer showed that the
pks12
mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the
pks12
gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the
pks12
mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus,
pks12
probably participates in DIM production and its expression is involved in pathogenesis.
The C-Terminal Domain of
Salmonella enterica
Serovar Typhimurium OmpA Is an Immunodominant Antigen in Mice but Appears To Be Only Partially Exposed on the Bacterial Cell Surface
Authors: Singh Shiva P. , Williams Yvonne U. , Miller Stephanie , Nikaido Hiroshi
Published: June 2003

We examined the way the major outer membrane protein OmpA of
Salmonella enterica
serovar Typhimurium is recognized by the mouse immune system, by raising a panel of 12 monoclonal antibodies (MAbs) against this protein. Interaction between OmpA and these MAbs is competitively inhibited with several-hundredfold dilutions of mouse polyclonal sera obtained by immunization with live or heat-killed whole cells, suggesting that OmpA is one of the immunodominant antigens of serovar Typhimurium. All of the MAbs were specific for an identical epitope(s) located on the C-terminal domain of OmpA, as indicated by the use of OmpA fragments generated by protease or cyanogen bromide treatment and by competitive inhibition enzyme-linked immunosorbent assay. This epitope was highly conserved within (but not outside) the family
Enterobacteriaceae
. The strong immunogenicity of this epitope was surprising because the C-terminal domain of OmpA, usually thought to be located in the periplasm, is not expected to be exposed on the bacterial cell surface. A MAb, however, reacted in a cytofluorometry assay more strongly with outer-membrane-permeabilized cells than with untreated cells, a result supporting the predominantly periplasmic localization of the epitope. Significant, though low-level, reactivity of intact cells nevertheless suggests that in some cells the C-terminal domain of OmpA is exposed on the surface, a result consistent with the proposal that OmpA can fold into one of the two alternate conformations.
Distribution and movement ofCaenorhabditis eleganson a thermal gradient
Authors: Yamada Yohko , Ohshima Yasumi
Published: June 2003

To analyze thermal responses of Caenorhabditis elegans in detail,distribution of a worm population and movement of individual worms were examined on a linear, reproducible and broad temperature gradient. Assay methods were improved compared with those reported previously to ensure good motility and dispersion of worms. Well-fed, wild-type worms distributed over a wide temperature range of up to 10°C, and, within this range, worms migrated in both directions of the gradient at similar frequencies without any specific response to the growth temperature in most cases. By contrast, worms migrated down the gradient if put in a region warmer than the warm boundary of distribution. The distribution range changed depending on the growth temperature and starvation, but active avoidance of a starvation temperature was not detected. These findings contradict previous hypotheses of taxis or migration to the growth temperature in association with food and instead indicate avoidance of a warm temperature. Our results favor a model for thermal response of C. elegans that postulates a single drive based on warm sensation rather than downward and upward drives in the physiological temperature range. Mutants in ttx-3, tax-2, tax-4 or egl-4genes showed abnormal thermal responses, suggesting that these genes are involved in warm avoidance. Laser ablation and gene expression studies suggest that AFD neurons are not important, and tax-4 expression in neurons other than AFD is required, for warm avoidance.
MHC class II engagement inhibits CD99‐induced apoptosis and up‐regulation of T cell receptor and MHC molecules in human thymocytes and T cell line
Authors: Kim Min Kyung , Choi Yoon-La , Kim Min Kyung , Kim Seok-Hyung , Choi Eun Young , Park Won Seo , Bae Young Mee , Woo Sang Kyu , Park Seong Hoe
Published: June 2003

Major histocompatibility complex (MHC) class II surface levels on thymocytes increase after CD99 ligation. The functional implication of the up‐regulated MHC class II was assessed by engaging MHC class II on CD99‐ligated cells. MHC class II engagement down‐modulated surface levels of T cell receptor and MHC molecules, and inhibited apoptosis of CD99‐ligated thymocytes and CEM tumor cells, antagonistic effects on the previously reported CD99 functions. The results were reproducible regardless of the order of ligation of MHC class II and CD99. We suggest that signaling via MHC class II on CD99‐engaged cells might be involved in the thymic maturation process by damping CD99 ligation effects.
Chondrocyte apoptosis induced by collagen degradation: Inhibition by caspase inhibitors and IGF‐1
Authors: Lo Marvin Y. , Kim Hubert T.
Published: June 2003

The main objective of this study was to test the effectiveness of candidate apoptosis inhibitors in limiting chondrocyte apoptosis induced by collagen degradation. Primary human chondrocytes were isolated from normal articular cartilage and grown in monolayer culture. Collagenase was added to the cells in the presence and absence of caspase inhibitors and insulin like growth factor (IGF)‐1. The amount of chondrocyte apoptosis was measured using an enzyme linked immunosorbent assay for nucleosomes, a specific and quantitative measure of apoptosis. Chondrocyte apoptosis was induced by collagenase treatment in both a time and dose dependent manner. The non‐selective caspase inhibitor Z‐VAD, the caspase‐3 selective inhibitor Z‐DEVD, and the growth factor insulin like growth factor (IGF)‐1 inhibited chondrocyte apoptosis induced by collagenase treatment. The caspase‐1 selective inhibitor Z‐YVAD also blocked chondrocyte apoptosis under these conditions, in contrast to previous studies where caspase‐1 inhibition failed to block apoptosis induced by agents such as the topoisomerase inhibitor camptothecin. These data demonstrate that the response of chondrocytes to caspase inhibition may be dependent upon the specific stimulus that initiates apoptosis. Furthermore, these findings suggest that multiple pathways involving both the initiation and execution of programmed cell death are potential targets for chondrocyte apoptosis inhibition therapy. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
ERBB-2 overexpression and cyclooxygenase-2 up-regulation in human cholangiocarcinoma and risk conditions
Authors: Endo Kanenori , Yoon Byung-IL , Pairojkul Chawalit , Demetris Anthony J. , Sirica Alphonse E.
Published: June 2003

Quantitative immunohistochemistry of ERBB-2 and MET receptor proteins and of cyclooxygenase 2 (COX-2) was undertaken to determine if there is a positive correlation between overexpression of either ERBB-2 or MET and up-regulation of COX-2 in human cholangiocarcinogenesis. ERBB-2, MET, and COX-2 immunoreactivities were measured in cancerous parenchyma of 71 archival cases of human cholangiocarcinoma (ChC) compared with hyperplastic small biliary ducts in surrounding nonneoplastic liver and with bile ducts of normal adult human liver. ERBB-2, MET, and COX-2 immunoreactivities were also assessed in both large and small hyperplastic biliary ducts (HBDs) in 27 archival cases of hepatolithiasis and 20 archival cases of primary sclerosing cholangitis (PSC), both of which are risk conditions for human cholangiocarcinogenesis. There was a strong positive correlation between increased ERBB-2, but not MET, and COX-2 immunoreactivity measured in the tumors and risk conditions. Enhanced immunoreactivity for ERBB-2 and COX-2 also correlated directly with tumor differentiation and was highest in well-differentiated tumors. Interestingly, some but not all cases of hepatolithiasis and most cases of PSC showed increased ERBB-2 and COX-2 immunostaining in the large but not small HBDs, whereas strong MET immunostaining was detected in both the large and small ducts. In conclusion, overexpression of ERBB-2 and COX-2 may herald an early carcinogenic event in the human hepatic biliary tract and one that is consistent with a frequent anatomic site of origin of the tumors. The results also suggest ERBB-2 and COX-2 as potentially important targets relevant to chemoprevention or adjunct therapy of ChC.
Teichoic Acids Are Not Required forStreptococcus pneumoniaeandStaphylococcus aureusCell Walls To Trigger the Release of Tumor Necrosis Factor by Peripheral Blood Monocytes
Authors: Majcherczyk P. A. , Rubli E. , Heumann D. , Glauser M. P. , Moreillon P.
Published: June 2003

In gram-negative bacteria, the outer membrane lipopolysaccharide is the main component triggering cytokine release from peripheral blood mononuclear cells (PBMCs). In gram-positive bacteria, purified walls also induce cytokine release, but stimulation requires 100 times more material. Gram-positive walls are complex megamolecules reassembling distinct structures. Only some of them might be inflammatory, whereas others are not. Teichoic acids (TA) are an important portion (≥50%) of gram-positive walls. TA directly interact with C3b of complement and the cellular receptor for platelet-activating factor. However, their contribution to wall-induced cytokine-release by PBMCs has not been studied in much detail. In contrast, their membrane-bound lipoteichoic acids (LTA) counterparts were shown to trigger inflammation and synergize with peptidoglycan (PGN) for releasing nitric oxide (NO). This raised the question as to whether TA are also inflammatory. We determined the release of tumor necrosis factor (TNF) by PBMCs exposed to a variety of TA-rich and TA-free wall fragments fromStreptocccus pneumoniaeandStaphylococcus aureus. TA-rich walls from both organisms induced measurable TNF release at concentrations of 1 μg/ml. Removal of wall-attached TA did not alter this activity. Moreover, purified pneumococcal and staphylococcal TA did not trigger TNF release at concentrations as high as ≥100 μg/ml. In contrast, purified LTA triggered TNF release at 1 μg/ml. PGN-stem peptide oligomers lacking TA or amino-sugars were highly active and triggered TNF release at concentrations as low as 0.01 μg/ml (P. A. Majcherczyk, H. Langen, et al., J. Biol. Chem. 274:12537-12543,1999). Thus, although TA is an important part of gram-positive walls, it did not participate to the TNF-releasing activity of PGN.
Control of the Oxidative Burst of Human Neutrophils by Staphylococcal Leukotoxins
Authors: Colin Didier A. , Monteil Henri
Published: June 2003

The ability of staphylococcal two-component leukotoxins to induce an oxidative burst and/or to prime human polymorphonuclear cells (PMNs) was studied by using spectrofluorometry or flow cytometry. At sublytic concentrations, the HlgA-HlgB, HlgA-LukF-PV, LukS-PV-LukF-PV, and HlgC-LukF-PV combinations of leukotoxins, but not the LukS-PV-HlgB and HlgC-HlgB combinations, were able to induce H
2
O
2
production similar to the H
2
O
2
production induced by 1 μM
N
-formyl-Met-Leu-Phe (fMLP). In addition, when added at sublytic concentrations, all of the leukotoxin combinations primed PMNs for H
2
O
2
production induced by fMLP. Leukotoxin activation was dependent on the presence of Ca
2+
and was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but not by
N
-methyl-
l
-arginine, an inhibitor of NO generation, which eliminates the possibility that NO plays a role in the action of leukotoxins. At higher concentrations, all leukotoxins inhibited H
2
O
2
production by PMNs activated by fMLP, phorbol 12-myristate 13-acetate (PMA), or the leukotoxins themselves. This inhibition was not related to the pore formation induced by leukotoxins. Intracellular release of H
2
O
2
induced by fMLP and PMA was not primed by leukotoxins but was inhibited. It seems that leukotoxin inhibition of H
2
O
2
release is independent of pore formation but secondary to an intracellular event, as yet unknown, triggered by leukotoxins.
Salmonella enterica
Serovar Typhimurium Expressing a Chromosomally Integrated Copy of the
Bacillus anthracis
Protective Antigen Gene Protects Mice against an Anthrax Spore Challenge
Authors: Garmory Helen S. , Titball Richard W. , Griffin Kate F. , Hahn Ulrike , Böhm Reinhard , Beyer Wolfgang
Published: June 2003

Protective immunity against infection with
Bacillus anthracis
is almost entirely based on a response to the protective antigen (PA), the binding moiety for the two other toxin components. We cloned the PA gene into an auxotrophic mutant of
Salmonella enterica
serovar Typhimurium as a fusion with the signal sequence of the hemolysin (Hly) A gene of
Escherichia coli
to allow the export of PA via the Hly export system. To stabilize the export cassette, it was also integrated into the chromosome of the live
Salmonella
carrier. When
S
.
enterica
serovar Typhimurium with the chromosomally integrated PA gene was given intravenously to A/J mice, they developed high levels of antibody to PA. These mice were protected against intraperitoneal challenge with 100 or 1,000 50% lethal doses of
B
.
anthracis
strain STI. This work contributes to the development of a
Salmonella
-based orally delivered anthrax vaccine.
The Delta Subunit of RNA Polymerase Is Required for Virulence of
Streptococcus agalactiae
Authors: Jones Amanda L. , Needham Rachel H. V. , Rubens Craig E.
Published: June 2003

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia, and meningitis in the United States despite the chemoprophylaxis strategies for preventing infection recommended by the Centers for Disease Control and Prevention. Using signature-tagged transposon mutagenesis to screen for novel virulence factors, we identified the
rpoE
gene as essential for development of sepsis in a neonatal rat model of GBS infection. An
rpoE
allelic replacement mutant displayed attenuated virulence in the sepsis model of infection identical to that of the transposon mutant, confirming linkage of the phenotype to the mutation in
rpoE
. The
rpoE
mutants also displayed increased sensitivity to killing in whole-blood bactericidal assays, which may explain the attenuated virulence. The mutants were otherwise phenotypically identical to the wild-type strain, including growth rate in plasma, indicating that a growth defect is not responsible for the attenuated virulence.
rpoE
is found only in gram-positive bacterial species and encodes the delta peptide, a subunit of RNA polymerase. Previous in vitro studies in other bacteria suggest that the delta peptide plays a role in maintaining transcriptional fidelity by blocking RNA polymerase binding at all but the strongest promoters, thereby inhibiting initiation of transcription. Despite the availability of
rpoE
mutants for several gram-positive bacterial species, a role for the peptide in vivo has not been defined, though it has been postulated that the delta peptide may be important for long-term survival in vitro or during growth phase transitions. Our data represent the first report of a phenotype relevant to virulence for
rpoE
mutants.
Differential Effect of p47phoxand gp91phoxDeficiency on the Course of Pneumococcal Meningitis
Authors: Schaper Manuela , Leib Stephen L. , Meli Damian N. , Brandes Ralf P. , Täuber Martin G. , Christen Stephan
Published: June 2003

Bacterial meningitis is a severe inflammatory disease of the central nervous system and is characterized by massive infiltration of granulocytes into the cerebrospinal fluid (CSF). To assess the role of NADPH oxidase-derived reactive oxygen species (ROS) in pneumococcal meningitis, mice deficient in either the gp91 subunit (essential for functioning of the phagocyte enzyme) or the p47 subunit (essential for functioning of homologous enzymes in nonphagocytic cells) were intracisternally infected with liveStreptococcus pneumoniae, and defined disease parameters were measured during the acute stage of infection. While none of the parameters measured (including CSF bacterial titers) were significantly different in gp91−/−and wild-type mice, the infection in p47−/−mice was associated with significantly increased inflammation of the subarachnoid and ventricular space, disruption of the blood-brain barrier, and the presence of interleukin-1β, tumor necrosis factor alpha, and matrix metalloproteinase 9 in the cortex. These changes were associated with ∼10-fold-higher CSF bacterial titers in p47−/−mice than in wild-type mice (P< 0.001). In contrast to infection with live bacteria, the inflammatory response, including CSF leukocytosis, was significantly attenuated in p47−/−mice (but not gp91−/−mice) challenged with a fixed number of heat-inactivated pneumococci. Impairment of the host defense appeared to be responsible for the higher bacterial titers in p47−/−mice. Therefore, these results indicate that ROS generated by a gp91-independent NADPH oxidase(s) are important for establishing an adequate inflammatory response to pneumococcal CSF infection.
Nuclear Factor κB Protects against Host Cell Apoptosis during
Rickettsia rickettsii
Infection by Inhibiting Activation of Apical and Effector Caspases and Maintaining Mitochondrial Integrity
Authors: Joshi Suresh G. , Francis Charles W. , Silverman David J. , Sahni Sanjeev K.
Published: June 2003

Apoptotic host cell death is a critical determinant in the progression of microbial infections and outcome of resultant diseases. The potentially fatal human infection caused by
Rickettsia rickettsii,
the etiologic agent of Rocky Mountain spotted fever, involves the vascular endothelium of various organ systems of the host. Earlier studies have shown that survival of endothelial cells (EC) during this infection depends on their ability to activate the transcription factor nuclear factor κB (NF-κB). Here, we investigated the involvement of caspase cascades and associated signaling pathways in regulation of host cell apoptosis by NF-κB. Infection of cultured human EC with
R. rickettsii
with simultaneous inhibition of NF-κB induced the activation of apical caspases 8 and 9 and also the executioner enzyme, caspase 3, whereas infection alone had no significant effect. Inhibition of either caspase-8 or caspase-9 with specific cell-permeating peptide inhibitors caused a significant decline in the extent of apoptosis, confirming their importance. The peak caspase-3 activity occurred at 12 h postinfection and led to cleavage of poly(ADP-ribose) polymerase, followed by DNA fragmentation and apoptosis. However, the activities of caspases 6 and 7, other important downstream executioners, remained unchanged. Caspase-9 activation was mediated through the mitochondrial pathway of apoptosis, as evidenced by loss of transmembrane potential and cytoplasmic release of cytochrome
c.
These findings suggest that activation of NF-κB is required for maintenance of mitochondrial integrity of host cells and protection against infection-induced apoptotic death by preventing activation of caspase-9- and caspase-8-mediated pathways. Targeted inhibition of NF-κB may therefore be exploited to enhance the clearance of infections with
R. rickettsii
and other intracellular pathogens with similar survival strategies.
Virulence Factors and Clonal Relationships among
Escherichia coli
Strains Isolated from Broiler Chickens with Cellulitis
Authors: de Brito Benito Guimarães , Gaziri Luiz Carlos J. , Vidotto Marilda C.
Published: June 2003

In this study, we compared
Escherichia coli
isolates from chickens with avian cellulitis with those from feces of healthy chickens. Cellulitis-derived strains presented phenotypic and genotypic characteristics of greater virulence than did the fecal isolates. Phylogenetic analysis by repetitive extragenic palindromic-PCR showed that, in agreement with their virulence characteristics, the cellulitis isolates form two clonal groups distinct from the fecal isolates.
Transforming Growth Factor α Binds to
Trypanosoma cruzi
Amastigotes To Induce Signaling and Cellular Proliferation
Authors: Alexander A. Deloris , Villalta Fernando , Lima Maria F.
Published: June 2003

Macrophages secrete transforming growth factor α (TGF-α) to trigger proliferation of cancer cells. Here, we report a new role for TGF-α in modulating the direct cellular proliferation of a parasitic protozoan,
Trypanosoma cruzi
. Amastigotes present two classes of receptors for TGF-α with different binding affinities.
125
I-TGF-α binding was competed by an excess of cold epidermal growth factor and TGF-α but not by an irrelevant molecule. Upon binding of TGF-α to amastigotes, the ligand is internalized, inducing trypanosome tyrosine phosphorylation of 90- and 87-kDa proteins and increasing DNA synthesis and proliferation of amastigotes. Furthermore, exposure of macrophages to TGF-α induced increased amastigote proliferation. These results describe a novel mechanism used by amastigotes to regulate their proliferation mediated by a TGF-α-dependent signal transduction pathway.
Exacerbation of Alcoholic Liver Injury by Enteral Endotoxin in Rats
Authors: Mathurin Philippe , Deng Qing-Gao , Keshavarzian Ali , Choudhary Sandeep , Holmes Earle W. , Tsukamoto Hidekazu
Published: June 2003

Increased gut permeability (leaky gut) and endotoxin–mediated Kupffer cell activation are proposed as the mechanisms of alcoholic liver injury. Although ethanol feeding is shown to sensitize the liver for injury induced by parental administration of lipopolysaccharide (LPS), how enteral LPS loading affects alcoholic liver injury is yet to be tested. The present study provides direct evidence for enhanced entrance to portal circulation of LPS enterally administered to the intragastric ethanol infusion model. Portal and systemic blood endotoxin levels increased to 43.0 ± 4.1 and 6.2 ± 4.3 pg/mL at 2 hours following enteral LPS administration (5 mg/kg) in alcohol–fed animals, while no such increases were observed in pair–fed controls. However, endotoxin levels in systemic blood of alcohol–fed rats were reduced to 0 to 1.5 pg/mL 16 hours after LPS administration. Weekly enteral administration of LPS to the model for 9 weeks exacerbated an increase in plasma alanine transaminase (ALT) levels (227 ± 75 vs. 140 ± 70;P< .01), mononuclear infiltration (25 ± 22 vs. 6.4 ± 4.4/10 mm2;P= .02), sinusoidal congestion, and spotty necrosis, and induced diffuse coagulative necrosis and centrilobular fibrosis in some animals. Reverse–transcription polymerase chain reaction (RT–PCR) analysis confirmed the LPS effect at the tissue level by demonstrating accentuated induction of tumor necrosis factor α (TNF–α) and Cox–2 mRNA. In conclusion, enteral LPS administration potentiates alcoholic liver necrosis, inflammation, and fibrosis despite efficient endotoxin clearance by the liver and mild systemic endotoxemia that occurs episodically following enteral LPS challenge.
A Study of Carboplatin–Coated Tube for the Unresectable Cholangiocarcinoma
Authors: Mezawa Shinichi , Homma Hisato , Sato Tsutomu , Doi Tadashi , Miyanishi Koji , Takada Koichi , Kukitsu Takehiro , Murase Kazuyuki , Yoshizaki Naoto , Takahashi Minoru , Sakamaki Sumio , Niitsu Yoshiro
Published: June 2003

Most cases of cholangiocarcinoma have reached an unresectable stage by the time they are discovered despite significant progress of diagnostic modalities. Many of these patients with obstructive jaundice are often treated by biliary drainage using stents to relieve the jaundice. However, the stent patency period is as short as 3 to 9 months because of tumor ingrowth or overgrowth, and mean survival is at most 12 months. Therefore, both continuous relief of obstructive jaundice and local control of the tumor are required in the treatment for advanced cholangiocarcinoma. In this investigation, we developed a new percutaneous transhepatic biliary drainage tube coated with carboplatin (carboplatin–coated tube; CCT). CCT continuously released a fixed amount of carboplatin for 4 weeks and showed an antitumor effect on human cholangiocarcinoma cell line HuCC–T1 in vitro. When CCT was embedded in subcutaneous tumor inoculated in nude mice, a significant reduction of tumor size with no apparent damage to normal adjacent tissue was observed. On the basis of these studies, 5 patients with inoperable cholangiocarcinoma were treated with CCT for 4 weeks. Overall efficacy rate of 5 patients with cholangiocarcinoma was 60% (partial response in 3 and no change in 2). No apparent side effect was observed in these patients. Thus, CCT may provide a new treatment modality for this disease. Randomized controlled trials comparing CCT therapy with palliative stenting are required to confirm these results.
Treatment of liver failure in rats with end-stage cirrhosis by transplantation of immortalized hepatocytes
Authors: Cai Jin , Ito Masahiro , Nagata Hideo , Westerman Karen A. , LaFleur Daryl , Chowdhury Jayanta Roy , Leboulch Philippe , Fox Ira J.
Published: June 2003

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, 35 immortalized rat hepatocyte clones were developed by transduction with the gene encoding the simian virus 40 T antigen (SV40Tag). The SV40Tag sequence and a suicide gene, herpes simplex virus thymidine kinase (HSV-tk), were flanked by loxP sequences so that they could be excised by Cre/lox recombination. When transplanted into the spleens of portacaval-shunted rats, 3 of the 35 immortalized hepatocyte clones prevented the development of hyperammonemia-induced hepatic encephalopathy. The protection was reversed by treatment with ganciclovir, which kills HSV-tk-expressing cells. Transplantation of alginate-encapsulated, immortalized hepatocytes into the spleens of cirrhotic rats resulted in significant improvement in prothrombin time, serum albumin and bilirubin levels, hepatic encephalopathy score, and duration of survival. The metabolic support provided by the immortalized cells equaled that observed after transplantation of primary rat hepatocytes. In conclusion, immortalized hepatocytes can function as well as primary hepatocytes following transplantation and can be engineered to contain safeguards that could make them clinically useful. Further investigation is warranted regarding the mechanisms of loss of mass or function of the transplanted hepatocytes over time and how the relatively few engrafted hepatocytes can ameliorate liver decompensation in cirrhosis.
Bioactivation of nitroglycerin and vasomotor response to nitric oxide are impaired in cirrhotic rat livers
Authors: Dudenhoefer Amy A. , Loureiro-Silva Maurício R. , Cadelina Gregory W. , Gupta Tarun , Groszmann Roberto J.
Published: June 2003

Nitroglycerin (NTG), a nitric oxide (NO) donor, has been shown to reduce portal pressure in cirrhotic patients. Using the in situ perfusion of normal and cirrhotic rat livers, we compared the vascular relaxation induced by either NTG or the spontaneous nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Normal and cirrhotic livers were perfused (40 mL/min, 37°C) with Krebs’ solution in a recirculating system. After preconstriction with methoxamine (10−4 mol/L), a dose-response study was performed using 6 cumulative doses of NTG or SNAP (10−7 to 3 × 10−5 mol/L). NOx (NO2
− + NO3
−) production in the perfusate was measured by chemiluminescence. Cirrhotic livers exhibited lower vasorelaxant responses, compared with normal livers, to both NTG (P < .0001) and SNAP (P = .0020). In normal livers, NTG and SNAP induced similar vasorelaxant responses (P = .44). In cirrhotic livers, NTG induced less vasorelaxation than SNAP (P < .0001). In the presence of NTG (P = .0045), but not SNAP (P = .99), NOx production in experiments with cirrhotic livers was lower than in experiments with normal livers. In conclusion, in cirrhotic rat livers, the vasorelaxant response to NTG is impaired owing to both a decreased metabolism of this NO donor and an inability of the hepatic vasculature to respond to NO.