bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemoglobin‐based Red Blood Cell Substitutes
Authors: Chang Thomas Ming Swi
Published: August 2004

Abstract:  Polyhemoglobin is already well into the final stages of clinical trials in humans with one approved for routine clinical use in South Africa. Conjugated hemoglobin is also in ongoing clinical trials. Meanwhile, recombinant Hb has been modified to modulate the effects of nitric oxide. Other systems contain antioxidant enzymes for those clinical applications that may have potential problems related to ischemia‐reperfusion injuries. Other developments are based on hemoglobin‐lipid vesicles and also the use of nanotechnology and biodegradable copolymers to prepare nanodimension artificial red blood cells containing hemoglobin and complex enzyme systems.
A Paraprofessional's View of Supervision
Authors: HALL ROSIE L.
Published: August 2004

The experience of a black paraprofessional community worker is presented in this paper with special emphasis on the problems of shifting to a family‐oriented point of view in her work. Problems associated with the supervisory relationship and with the worker's use of herself as a therapeutic person are discussed as well.
Structured Separation with Counseling: A Therapeutic Approach for Couples in Conflict
Authors: TOOMIM MARJORIE KAWIN
Published: August 2004

A three‐month trial separation with counseling is suggested as a form of crisis intervention for couples in conflict. The purpose of the counseling is to help the marital partners understand their relationship, resolve their conflicts, decide whether their future relationship will be together or apart, and grow through the separation process. A one‐year follow‐up was conducted with eighteen couples who completed structured separation with counseling.
Family Interaction as a Function of Experimental Task
Authors: JACOB THEODORE , DAVIS JOHN
Published: August 2004

To assess consistency family interaction across different experimental tasks, the present study evaluated family patterns of talking and interrupting, as elicited from discussions of a Plan Something Together Task, a set of TAT cards, and an Unrevealed Difference Questionnaire. With few exceptions, data analyses indicated that such family patterns are not altered significantly as a function of experimental tasks and, therefore, suggest considerable interactional stability across differing contexts. Characteristics of the present study (as well as related investigations of task effects) are discussed in terms of implications for future research efforts.
Perception of Cognitive Ability in Families of Adolescents
Authors: SHAPIRO RODNEY J. , FISHER LAWRENCE , GAYTON WILLIAM F.
Published: August 2004

The families of 20 hospitalized adolescents were compared with a control group of 20 non‐pathological families on the accuracy with which family members could predict each others' performance on two tests of intellectual ability. Accuracy of prediction was measured by the discrepancies between actual scores obtained by each family member and the scores other family members predicted he or she would obtain. The control and experimental groups were found to differ significantly on socioeconomic status and intellectual ability, and when these variables were controlled, a covariance analysis revealed no significant difference between the groups on accuracy of predictions. The results suggest that variables other than psychopathology must be taken into account in explaining apparent differences between healthy and disturbed families on measurements of interpersonal judgments. Implications for research and family therapy are discussed.
“All in the Family” as a Mirror of Contemporary American Culture
Authors: STEIN HOWARD F.
Published: August 2004

The television series “All in the Family” is discussed as paradigmatic of contemporary American culture. By exploring the dramas the members of the Bunker household enact, the images and identities they project, and those projected upon them, the dynamics of American culture are delineated. The analysis pursues such dimensions as interpersonal process, generational conflict, identity structure, value conflict, and cultural patterning.
Matrix metalloproteinase‐1 and tissue inhibitor of metalloproteinase‐1 production in human gingival fibroblasts: the role of protein kinase C
Authors: Domeij H. , Modéer T. , Yucel‐Lindberg T.
Published: August 2004

Background:  Matrix metalloproteinase‐1 (MMP‐1) plays an important role in tissue remodelling and in the pathology of inflammatory diseases including periodontitis. The activity of MMP‐1 is firmly controlled by the endogenous tissue inhibitor of metalloproteinase‐1 (TIMP‐1).

Objective:  The aim of the study was to investigate the production and regulation of MMP‐1 and TIMP‐1 with special regards to the enzyme protein kinase C (PKC) in human gingival fibroblasts.

Methods:  Gingival fibroblasts were treated with substances related to PKC such as phorbol 12‐myristate 13‐acetate (PMA), interleukin‐1β, Ca2+‐ionophore A231817 and inhibitors of PKC, p38 mitogen‐activated protein kinase (p38 MAPK) and tyrosine kinase.

Results:  The PKC activator PMA stimulated the production of MMP‐1 and TIMP‐1 at both the transcriptional and the translational level. The production of MMP‐1 and TIMP‐1 stimulated by PMA was abolished by the PKC inhibitor bisindolylmaleimide. Treatment of the cells with interleukin‐1β or A23187 synergistically increased the stimulatory effect of PMA on MMP‐1 production. In contrast, TIMP‐1 production was unaffected by interleukin‐1β and reduced by A23187. Tyrosine kinase inhibitor herbimycin A reduced MMP‐1 production induced by PMA, whereas the p38 MAPK‐inhibitor SB 203580 synergistically increased the stimulatory effect of PMA on both MMP‐1 and TIMP‐1 production.

Conclusion:  The present study shows that MMP‐1 and TIMP‐1 production is regulated differently by interleukin‐1β and calcium in human gingival fibroblasts and that this difference is markedly amplified in the presence of the PKC‐activator PMA. Taken together, the discrepancy in the production of MMP‐1 and TIMP‐1 in gingival fibroblasts may contribute to tissue destruction in periodontal diseases.
γ‐Interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts
Authors: Mochizuki S. , Kobayashi M. , Suzuki T. , Oikawa A. , Koseki T. , Nishihara T. , Hasegawa K.
Published: August 2004

Objectives:  CD14, toll‐like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts.

Materials and methods:  Following treatment with either interleukin‐1β, tumor necrosis factor‐α (TNF‐α) or γ‐interferon (IFN‐γ), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN‐γ, cells were pre‐incubated with either anti‐CD14 antibody MY4 or anti‐TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen‐activated protein (MAP) kinases and IκBα was examined by western blotting, and production of interleukin‐6 and interleukin‐8 was measured by their respective enzyme‐linked immunosorbent assay (ELISA) kits.

Results:  IFN‐γ stimulated expression of mCD14, whereas ‐1β and TNF‐α did not. Expression of MyD88 but not TLR4 was also enhanced by IFN‐γ. The lipopolysaccharide activated MAP kinases, such as extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK) and p38, and IκBα and stimulated production of interleukin‐6 and interleukin‐8. The lipopolysaccharide‐stimulated interleukin‐6 and interleukin‐8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN‐γ augmented the following activation of MAP kinases and IκBα and production of interleukin‐6 and interleukin‐8 in response to the lipopolysaccharide.

Conclusions:  These results suggest that the augmentation by IFN‐γ of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IκBα and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up‐regulation of CD14 and MyD88 expression.
Critical steps in electronic volume quantification of gingival crevicular fluid: the potential impact of evaporation, fluid retention, local conditions and repeated measurements
Authors: Tözüm Tolga F. , Hatipoğlu Hasan , Yamalik Nermin , Gürsel Mihtikar , Alptekin Nilgün Ö. , Ataoğlu Tamer , Marakoğlu Ismail , Gürsoy Ulvi K. , Eratalay Kenan
Published: August 2004

Background and objectives:  Various methodological factors may operate during clinical gingival crevicular fluid (GCF) sampling, volume quantification or subsequent laboratory analysis. For precise volume quantification, specific concern for generation and maintenance of a reliable calibration curve, the potential risk of GCF loss as a result of evaporation or fluid retention on actual volume and the impact of local conditions is needed because each of these factors may act as a source of subsequent volumetric distortions. Thus, the present study aimed to analyse the impact of sample transfer time on the rate of evaporation and the possibility of fluid retention, and the impact of local conditions and number of replicated measurements on the reliability of calibration data.

Materials and methods:  To analyse evaporative errors, standardized Periopaper® strips provided with known test volumes (0.1 µl, 0.2 µl, 0.5 µl and 0.6 µl) were transferred to Periotron 8000® with different time intervals (immediately, 5 s, 30 s and 60 s). For fluid retention, after quantifying the actual volume of the strips provided with known volumes (0.1 µl and 0.6 µl) of two test fluids, a second set of measurements was performed using dry strips. To determine the impact of local conditions (temperature and humidity) and the validity of 3, 5 and 20 replications (0.0–0.6 µl with 0.1‐µl increments) on device calibration for 20°C and 25°C, electronic readings were obtained from three devices at three different locations. Differences in volumetric data in each experimental design were statistically analysed.

Results:  No significant fluid loss was observed within 5 s, but evaporation clearly led to volumetric distortions with extending transfer times (30 s or 60 s) (p < 0.05). Measurable amounts of fluid retention were found for both volumes and both test fluids, but not with identical patterns. Local conditions resulted in unique calibration data for each test volume and for each device. Although a 5°C increase generally provided higher readings, this was not observed for all devices at all volumes. Additional replicates (n = 5 or n = 20) did not seem to add any further reliability to the triplicate scores for the given test volumes.

Conclusion:  The findings of the present study confirm the reliability of triplicate readings, and uniqueness of each device and electronic data and the distinct impact of local environmental conditions on the generation/maintenance of calibration scores for each particular device. Furthermore, they underline time‐dependent evaporation and fluid retention as additional technical concerns and once again highlight the importance of methodological standardization of the electronic volume quantification process.
How New Is the New Economy?
Authors: Madrick Jeff
Published: August 2004

The strong economic performance of the past few years has led many observers to claim that hard evidence for a new economy now exists. In fact, says the author, we need many more years of evidence to make such a claim. Any economic recession soon will quickly reverse what gains in wages have been made.
“Never a Fee!”
Authors: Gonos George
Published: August 2004

Historical and legal research challenges the staffing industry's claim that it charges “no fees” to workers, and provides scholarly support for the spreading “right‐to‐know” movement among temps and contract workers for the disclosure of hidden, and often exorbitant, agency markups.
The Past and Future Contradictions and Challenges of Labor Leadership
Authors: Dubofsky Melvyn
Published: August 2004

Labor leaders of the future face the same dilemmas as those of the past: how to build large, militant unions while avoiding their own co‐optation by the establishment.
How the Internet Is Changing Unions
Authors: Lee Eric
Published: August 2004

Labor has drawn on the Internet to build cross‐border worker solidarity to counter management efforts against strikes and break union organizing efforts. This article examines how the Internet has already been used to labor's benefit in two key struggles.
The Teamsters' Overnite Strike
Authors: Slaughter Jane
Published: August 2004

In their drive to organize the largest nonunion trucker, the Teamsters called a strike that only a minority of drivers are honoring. Some members say President Hoffa may have jumped the gun.
The Once and Future History of Steady Work
Authors: Costello Tim
Published: August 2004

Workers have fought for over a century to create steady full‐time jobs. However, corporate restructuring in the last two decades has engendered the growth of unsteady work, which today is the fastest‐growing segment of the labor market. The author prescribes a new program to draw public attention to the inequities of contingent work and build a campaign to create steady jobs through popular and legislative action.
Toward a New Labor Rights Movement
Authors: Pope James , Kellman Peter , Bruno Ed
Published: August 2004

Workers are not entitled to the rights and privileges that are normally enjoyed by U.S. citizens. The writers argue that in today's workplace, workers are forced to abandon their constitutional rights in order to earn their living, and thus are subject to the tyranny of the corporation. Since legislative reform has proved a losing strategy, the writers propose a remedy of extending constitutional rights into the workplace.
Functional bradykinin B1 receptors are expressed in nociceptive neurones and are upregulated by the neurotrophin GDNF
Authors: Vellani Vittorio , Zachrisson Olof , McNaughton Peter A.
Published: August 2004

Bradykinin (BK) has long been recognized as an important mediator of pain and inflammation. In normal tissue bradykinin causes an acute sensation of pain by an action at B2 receptors, but in inflamed tissue the pharmacology of the response changes to that of B1 receptors. Attempts to demonstrate the presence of functional B1 receptors in sensory neurones have failed, however, and the actions of B1 agonists have therefore been presumed to be indirect. Here we show that specific B1 receptor activation causes translocation of the ɛ isoform of protein kinase C (PKC‐ɛ) to the membrane of a small fraction of freshly isolated sensory neurones from rats and mice. The proportion of neurones in which PKC‐ɛ translocation was observed increased to around 20% of neurones after 3 days in culture with the neurotrophins glial cell line derived neurotrophic factor (GDNF) and neurturin, but not with nerve growth factor (NGF). Using in situ hybridization we found that the proportion of neurones expressing B1 mRNA increased from close to zero to 20.4% after 8 h culture in GDNF. Neurones expressing functional B1 receptors were negative for the neuropeptides CGRP and substance P, but most expressed functional TRPV1 receptors for capsaicin (60%) and bound the lectin IB4 (68%), both markers characteristic of nociceptors. B1 activation enhanced the heat‐activated membrane current ∼3‐fold, and the enhancement was much more prolonged than was the case with B2 activation, consistent with a role for B1 receptors in sustained pain. We conclude that GDNF and neurturin potently upregulate functional B1 receptor expression in small non‐peptidergic nociceptive neurones.
Regulation of the hyperpolarization‐activated cationic current Ih in mouse hippocampal pyramidal neurones by vitronectin, a component of extracellular matrix
Authors: Vasilyev Dmitry V. , Barish Michael E.
Published: August 2004

Because the hyperpolarization‐activated cation‐selective current Ih makes important contributions to neural excitability, we examined its long‐term regulation by vitronectin, an extracellular matrix component commonly elevated at injury sites and detected immunochemically in activated microglia. Focusing on mouse hippocampal pyramidal neurones in organotypic slice cultures established at postnatal day 0 or 1 and examined after 3–4 days in vitro, we observed differences in the amplitude and activation rate of Ih between neurones in naive and vitronectin‐exposed slices (10 μg ml−1 added to serum‐free medium), and between neurones in slices derived from wild‐type and vitronectin‐deficient mice. The potassium inward rectifier IK(ir), activated at similar voltages to Ih, was not affected by vitronectin. In CA1, differences in Ih amplitude primarily reflected changes in maximum conductance (Gmax): a 23.3% increase to 3.18 ± 0.64 nS from 2.58 ± 0.96 nS (P < 0.05) in vitronectin‐exposed neurones, and a 17.9% decrease to 2.24 ± 0.26 nS from 2.73 ± 0.64 nS (P < 0.05) in neurones from vitronectin‐deficient slices. The voltage of one‐half maximum activation (V½) was not significantly affected by vitronectin exposure (−78.1 ± 2.3 mV versus−80.0 ± 4.9 mV in naive neurones; P > 0.05) or vitronectin deficiency (−83.8 ± 3.1 mV versus−82.0 ± 2.9 mV in wild‐type neurones; P > 0.05). In CA3 neurones, changes in Ih reflected differences in both Gmax and V½: in vitronectin‐exposed neurones there was a 35.4% increase in Gmax to 1.30 ± 0.49 nS from 0.96 ± 0.26 nS (P < 0.01), and a +3.0 mV shift in V½ to −89.8 mV from −92.8 mV (P < 0.05). The time course of Ih activation could be fitted by the sum of two exponential functions, fast and slow. In both CA1 and CA3 neurones the fast component amplitude was preferentially sensitive to vitronectin, with its relatively larger contribution to total current in vitronectin‐exposed cells contributing to the acceleration of Ih activation. Further, HCN1 immunoreactivity appeared elevated in vitronectin‐exposed slices, while HCN2 levels appeared unaltered. We suggest that vitronectin‐stimulated increases in Ih may potentially affect excitability under pathological conditions.
Fusion‐independent expression of functional ACh receptors in mouse mesoangioblast stem cells contacting muscle cells
Authors: Grassi Francesca , Pagani Francesca , Spinelli Gabriele , Angelis Luciana De , Cossu Giulio , Eusebi Fabrizio
Published: August 2004

Mesoangioblasts are vessel‐associated fetal stem cells that can be induced to differentiate into skeletal muscle, both in vitro and in vivo. Whether this is due to fusion or to transdifferentiation into bona fide satellite cells is still an open question, for mesoangioblasts as well as for other types of stem cells. The early steps of satellite cell myogenic differentiation involve MyoD activation, membrane hyperpolarization and the appearance of ACh sensitivity and gap junctional communication. If mesoangioblasts differentiate into satellite cells, these characteristics should be observed in stem cells prior to fusion into multinucleated myotubes. We have investigated the functional properties acquired by mononucleated green fluorescent protein (GFP)‐positive mesoangioblasts co‐cultured with differentiating C2C12 myogenic cells, using the patch‐clamp technique. Mesoangioblasts whose membrane contacted myogenic cells developed a hyperpolarized membrane resting potential and ACh‐evoked current responses. Dye and electrical coupling was observed among mesoangioblasts but not between mesoangioblasts and myotubes. Mouse MyoD was detected by RT‐PCR both in single, mononucleated mesoangioblasts co‐cultured with C2C12 myotubes and in the total mRNA from mouse mesoangioblasts co‐cultured with human myotubes, but not in human myotubes or stem cells cultured in isolation. In conclusion, when co‐cultured with muscle cells, mesoangioblasts acquire many of the functional characteristics of differentiating satellite cells in the absence of cell fusion, strongly indicating that these stem cells undergo transdifferentiation into satellite cells, when exposed to a myogenic environment.
Chronic intermittent hypoxia enhances cat chemosensory and ventilatory responses to hypoxia
Authors: Rey Sergio , Rio Rodrigo Del , Alcayaga Julio , Iturriaga Rodrigo
Published: August 2004

The carotid body (CB) chemoreceptors may play an important role in the enhanced hypoxic ventilatory response induced by chronic intermittent hypoxia (CIH). We studied the effects of cyclic hypoxic episodes of short duration on cat cardiorespiratory reflexes, heart rate variability, and CB chemosensory activity. Cats were exposed to cyclic hypoxic episodes (P∼ 75 Torr) repeated during 8 h for 2–4 days. Cats were anaesthetized with sodium pentobarbitone (40 mg kg−1i.p., followed by 8–12 mg i.v.), and ventilatory and cardiovascular responses to NaCN (0.1–100 μg kg−1i.v.) and several isocapnic levels of oxygen (P∼ 20–740 Torr) were studied. After studying the reflex responses, we recorded the CB chemosensory responses induced by the same stimuli. Results showed that CIH for 4 days selectively enhanced cat CB ventilatory (VT and VI) responses to hypoxia, while responses to NaCN remained largely unchanged. Similarly, basal CB discharges and responses to acute hypoxia (P < 100 Torr) were larger in CIH than in control cats, without modification of the responses to NaCN. Exposure to CIH did not increase basal arterial pressure, heart rate, or their changes induced by acute hypoxia or hyperoxia. However, the spectral analysis of heart rate variability of CIH cats showed a marked increase of the low‐/high‐frequency ratio and an increase of the power spectral distribution of low frequencies of heart rate variability. Thus, the enhanced CB reactivity to hypoxia may contribute to the augmented ventilatory response to hypoxia, as well as to modified heart rate variability due to early changes in autonomic activity.
Intramembrane Proteolysis: Theme and Variations
Authors: Wolfe Michael S. , Kopan Raphael
Published: August 2004

Proteases that reside in cellular membranes apparently wield water to hydrolyze the peptide bonds of substrates despite their water-excluding environment. Although these intramembrane proteases bear little or no sequence resemblance to classical water-soluble proteases, they have ostensibly converged on similar hydrolytic mechanisms. Identification of essential amino acid residues of these proteases suggests that they use residue combinations for catalysis in the same way as their soluble cousins. In contrast to classical proteases, however, the catalytic residues of intramembrane proteases lie within predicted hydrophobic transmembrane domains. Elucidating the biological functions of intramembrane proteases, identifying their substrates, and understanding how they hydrolyze peptide bonds within membranes will shed light on the ways these proteases regulate crucial biological processes and contribute to disease.
Regions of Strong Coupling Between Soil Moisture and Precipitation
Authors: Koster Randal D. , Dirmeyer Paul A. , Guo Zhichang , Bonan Gordon , Chan Edmond , Cox Peter , Gordon C. T. , Kanae Shinjiro , Kowalczyk Eva , Lawrence David , Liu Ping , Lu Cheng-Hsuan , Malyshev Sergey , McAvaney Bryant , Mitchell Ken , Mocko David , Oki Taikan , Oleson Keith , Pitman Andrew , Sud Y. C. , Taylor Christopher M. , Verseghy Diana , Vasic Ratko , Xue Yongkang , Yamada Tomohito
Published: August 2004

Previous estimates of land-atmosphere interaction (the impact of soil moisture on precipitation) have been limited by a lack of observational data and by the model dependence of computational estimates. To counter the second limitation, a dozen climate-modeling groups have recently performed the same highly controlled numerical experiment as part of a coordinated comparison project. This allows a multimodel estimation of the regions on Earth where precipitation is affected by soil moisture anomalies during Northern Hemisphere summer. Potential benefits of this estimation may include improved seasonal rainfall forecasts.
Crystal Structure of a Shark Single-Domain Antibody V Region in Complex with Lysozyme
Authors: Stanfield Robyn L. , Dooley Helen , Flajnik Martin F. , Wilson Ian A.
Published: August 2004

Cartilaginous fish are the phylogenetically oldest living organisms known to possess components of the vertebrate adaptive immune system. Key to their immune response are heavy-chain, homodimeric immunoglobulins called new antigen receptors (IgNARs), in which the variable (V) domains recognize antigens with only a single immunoglobulin domain, akin to camelid heavy-chain V domains. The 1.45 angstrom resolution crystal structure of the type I IgNAR V domain in complex with hen egg-white lysozyme (HEL) reveals a minimal antigen-binding domain that contains only two of the three conventional complementarity-determining regions but still binds HEL with nanomolar affinity by means of a binding interface comparable in size to conventional antibodies.
DNA-Templated Organic Synthesis and Selection of a Library of Macrocycles
Authors: Gartner Zev J. , Tse Brian N. , Grubina Rozalina , Doyon Jeffrey B. , Snyder Thomas M. , Liu David R.
Published: August 2004

The translation of nucleic acid libraries into corresponding synthetic compounds would enable selection and amplification principles to be applied to man-made molecules. We used multistep DNA-templated organic synthesis to translate libraries of DNA sequences, each containing three “codons,” into libraries of sequence-programmed synthetic small-molecule macrocycles. The resulting DNA-macrocycle conjugates were subjected to in vitro selections for protein affinity. The identity of a single macrocycle possessing known target protein affinity was inferred through the sequence of the amplified DNA template surviving the selection. This work represents the translation, selection, and amplification of libraries of nucleic acids encoding synthetic small molecules rather than biological macromolecules.
Streptococcus pneumoniae-Induced Caspase 6-Dependent Apoptosis in Lung Epithelium
Authors: Schmeck Bernd , Gross Ralph , N′Guessan Phillipe Dje , Hocke Andreas C. , Hammerschmidt Sven , Mitchell Tim J. , Rosseau Simone , Suttorp Norbert , Hippenstiel Stefan
Published: August 2004

Streptococcus pneumoniaeis the major pathogen of community-acquired pneumonia and one of the most common causes of death due to infectious diseases in industrialized countries. Lung epithelium lines the airways and constitutes the first line of innate defense against respiratory pathogens. Little is known about the molecular interaction of pneumococci with lung epithelial cells. Apoptosis of lung epithelium is involved in some bacterial lung infections. In this study different pneumococcal strains specifically induced either apoptotic or necrotic death of human alveolar and bronchial epithelial cells. Pneumococcus-induced apoptosis did not depend on the virulence factors pneumolysin and H2O2. Apoptotic cells showed increased activity of caspases 6, 8, and 9 but not increased activity of caspase 3. Moreover, programmed cell death could be strongly reduced by a caspase 6 inhibitor and a pan-caspase inhibitor. Inhibitors of calpain and chymotrypsin- and trypsin-like proteases also reduced pneumococcus-induced apoptosis. Furthermore, pneumococcus-infected human alveolar epithelial cells showed Bid cleavage and reduced levels of Bcl2 and Bax. Overexpression of Bcl2 in these cells reduced apoptosis significantly. Thus, pneumococci induced apoptosis of human alveolar and bronchial epithelial cells. Programmed cell death was executed by caspase 6 and noncaspase proteases, but not by caspase 3, and could be blocked by overexpression of Bcl2.
Global Cell Surface Conformational Shift Mediated by a
Candida albicans
Adhesin
Authors: Rauceo Jason M. , Gaur Nand K. , Lee Kyeng-Gea , Edwards John E. , Klotz Stephen A. , Lipke Peter N.
Published: August 2004

Candida albicans
maintains both commensal and pathogenic states in humans. Both states are dependent on cell surface-expressed adhesins, including those of the Als family. Heterologous expression of Als5p at the surface of
Saccharomyces cerevisiae
results in Als5p-mediated adhesion to various ligands, followed by formation of multicellular aggregates. Following adhesion of one region of the cell to fibronectin-coated beads, the entire surface of the cells became competent to mediate cell-cell aggregation. Aggregates formed in the presence of metabolic inhibitors or signal transduction inhibitors but were reduced in the presence of 8-anilino-1-naphthalene-sulfonic acid (ANS) or Congo Red (CR), perturbants that inhibit protein structural transitions. These perturbants also inhibited aggregation of
C. albicans
. An increase in ANS fluorescence, which accompanied Als-dependent cellular adhesion, indicated an increase in cell surface hydrophobicity. In addition,
C. albicans
and Als5p-expressing
S. cerevisiae
showed an aggregation-induced birefringence indicative of order on the cell surface. The increase in birefringence did not occur in the presence of the aggregation disruptants ANS and CR. These results suggest a model for Als5p-mediated aggregation in which an adhesion-triggered change in the conformation of Als5p propagates around the cell surface, forming ordered aggregation-competent regions.
Treatment of Mice with the Neutrophil-Depleting Antibody RB6-8C5 Results in Early Development of Experimental Lyme Arthritis via the Recruitment of Gr-1−Polymorphonuclear Leukocyte-Like Cells
Authors: Brown Charles R. , Blaho Victoria A. , Loiacono Christie M.
Published: August 2004

Recently, we demonstrated that blocking the entry of neutrophils intoBorrelia burgdorferi-infected joints in mice deficient in the chemokine receptor CXCR2 prevented the development of experimental Lyme arthritis. Neutrophils were marginalized in blood vessels at the site of infection but could not enter the joint tissue. In the present study, we treated both genetically arthritis-resistant DBA/2J (DBA) and arthritis-susceptible C3H/HeJ (C3H) mice with the neutrophil-depleting monoclonal antibody RB6-8C5 (RB6) to determine the effect on arthritis development. Surprisingly, both DBA and C3H mice treated with RB6 developed arthritis at 1 week postinfection, approximately 1 week earlier than the control-treated C3H mice. The early development of arthritis in the RB6-treated mice was accompanied by an influx into the joints of cells with ring-shaped polymorphonuclear leukocyte (PMN) cell morphology that were negative for the Gr-1 neutrophil maturation marker. RB6 treatment of mice also resulted in increased numbers ofB. burgdorfericells in the joints at 7 days postinfection and earlier expression of the chemokines KC and monocyte chemoattractant protein 1 in the joints compared to control-treated animals. Together, these results suggest that recruitment of neutrophils or PMN-like cells into an infected joint is a key requirement for Lyme arthritis development and that altered recruitment of these cells into the joints of arthritis-resistant mice can exacerbate the development of pathology.
CD4+T Cells in the Pathogenesis of Murine Ocular Toxoplasmosis
Authors: Lu Fangli , Huang Shiguang , Kasper Lloyd H.
Published: August 2004

The role of CD4+T cells in the pathogenesis of ocular toxoplasmosis was investigated in murine models utilizing inbred C57BL/6 mice deficient either in CD4+, CD8+, or B cells (μMT). Severe necrosis and inflammation with replicating parasites were observed in the eyes of control mice after primary ocular infection, and near-normal histology with few tachyzoites was observed in the eyes of mice immunized intraperitoneally with the avirulent ts-4 strain followed by intraocular challenge with the RH strain ofToxoplasma gondii. In contrast, mild inflammation without evidence of necrosis associated with increased parasite burdens were observed in the eyes of CD4 knockout (KO) mice after both primary ocular infection and challenge with RH tachyzoites. CD8 KO mice, as well as μMT mice, demonstrated increased ocular necrosis in response to either primary ocular infection or challenge. The parasite burden was increased in the eyes of both CD8 KO and μMT mice in which the parasite load was even higher. As expected, there were no increases in the levels of immunoglobulin G in serum or aqueous humor in μMT mice, and there was no increase in the levels of gamma interferon and tumor necrosis factor alpha in the sera of CD4 KO mice after both infection and challenge. These results suggest that the ocular inflammatory response to the parasite is mediated primarily by the CD4+-T-cell response. CD8+T cells and B cells may play an important role in limiting tachyzoite proliferation in the eyes. Mice deficient in CD8+CD4+T cells or B cells exhibit diminished vaccine-induced resistance and increased ocular parasite burden after challenge.
The Response Regulator PhoP of
Yersinia pseudotuberculosis
Is Important for Replication in Macrophages and for Virulence
Authors: Grabenstein Jens P. , Marceau Michael , Pujol Céline , Simonet Michel , Bliska James B.
Published: August 2004

Yersinia pestis
and
Yersinia pseudotuberculosis
are closely related facultative intracellular pathogens. The response regulator PhoP was previously shown to be important for
Y. pestis
survival in macrophages and for virulence in a murine bubonic plague infection assay. Here the importance of PhoP for
Y. pseudotuberculosis
pathogenesis was investigated.
Y. pseudotuberculosis phoP
mutants were unable to replicate in low-Mg
2+
medium or in macrophages.

phoP
+
Y. pseudotuberculosis

strains initiated replication in macrophages after a lag period of ∼5 h, as shown by fluorescence microscopy and viable count assays.
Y. pseudotuberculosis phoP
mutants died at a low rate in macrophages; there was no decrease in viability over the first 5 h of infection, and there was a 10-fold decrease in viability between 5 and 24 h of infection. Trafficking of phagosomes containing

phoP
+

or
phoP
mutant
Y. pseudotuberculosis
was studied by using immunofluorescence microscopy and cathepsin D as a marker for lysosomes. Phagosomes containing
phoP
mutant
Y. pseudotuberculosis
acquired cathepsin D at a higher rate than phagosomes containing

phoP
+

bacteria. However, the increased rate of marker acquisition for phagosomes containing mutant bacteria was only evident ∼5 h after infection, suggesting that
phoP
mutants are able to retard phagosome maturation during the lag phase of intracellular growth. The results obtained with a
Y. pestis phoP
mutant were similar to those described above, except that the rates of intracellular killing and trafficking to cathepsin D-positive vacuoles were significantly higher. A
Y. pseudotuberculosis phoP
mutant was 100-fold less virulent than the wild-type strain in a murine intestinal infection model, suggesting that survival and replication in macrophages are important for
Y. pseudotuberculosis
pathogenesis.
Cryptococcal Yeast Cells Invade the Central Nervous System via Transcellular Penetration of the Blood-Brain Barrier
Authors: Chang Yun C. , Stins Monique F. , McCaffery Michael J. , Miller Georgina F. , Pare Dan R. , Dam Tapen , Paul-Satyasee Maneesh , Kim Kwang Sik , Kwon-Chung Kyung J.
Published: August 2004

Cryptococcal meningoencephalitis develops as a result of hematogenous dissemination of inhaled
Cryptococcus neoformans
from the lung to the brain. The mechanism(s) by which
C. neoformans
crosses the blood-brain barrier (BBB) is a key unresolved issue in cryptococcosis. We used both an in vivo mouse model and an in vitro model of the human BBB to investigate the cryptococcal association with and traversal of the BBB. Exposure of human brain microvascular endothelial cells (HBMEC) to
C. neoformans
triggered the formation of microvillus-like membrane protrusions within 15 to 30 min. Yeast cells of
C. neoformans
adhered to and were internalized by the HBMEC, and they crossed the HBMEC monolayers via a transcellular pathway without affecting the monolayer integrity. The histopathology of mouse brains obtained after intravenous injection of
C. neoformans
showed that the yeast cells either were associated with endothelial cells or escaped from the brain capillary vessels into the neuropil by 3 h.
C. neoformans
was found in the brain parenchyma away from the vessels by 22 h. Association of
C. neoformans
with the choroid plexus, however, was not detected during up to 10 days of observation. Our findings indicate that
C. neoformans
cells invade the central nervous system by transcellular crossing of the endothelium of the BBB.
Role of Antibiotics and Fungal Microbiota in Driving Pulmonary Allergic Responses
Authors: Noverr Mairi C. , Noggle Rachael M. , Toews Galen B. , Huffnagle Gary B.
Published: August 2004

Over the past four decades, there has been a significant increase in allergy and asthma in westernized countries, which correlates with alterations in fecal microbiota (microflora) and widespread use of antibiotics (the “hygiene hypothesis”). Antibiotics also lead to overgrowth of the yeastCandida albicans, which can secrete potent prostaglandin-like immune response modulators. We have developed a mouse model of antibiotic-induced microbiota disruption that includes stable increases in gastrointestinal (GI) enteric bacteria and GICandidalevels with no introduction of microbes into the lungs. Mice are treated for 5 days with cefoperazone in the drinking water, followed by a single oral gavage ofC. albicans. This results in alterations of GI bacterial populations and increased yeast numbers in the GI microbiota for at least 2 to 3 weeks and can drive the development of a CD4 T-cell-mediated allergic airway response to subsequent mold spore (Aspergillus fumigatus) exposure in immunocompetent mice without previous systemic antigen priming. The allergic response in the lungs is characterized by increased levels of eosinophils, mast cells, interleukin-5 (IL-5), IL-13, gamma interferon, immunoglobulin E, and mucus-secreting cells. In the absence of antibiotics, mice exposed toAspergillusspores do not develop an allergic response in the airways. This study provides the first experimental evidence to support a role for antibiotics and fungal microbiota in promoting the development of allergic airway disease. In addition, these studies also highlight the concept that events in distal mucosal sites such as the GI tract can play an important role in regulating immune responses in the lungs.
Mycoplasma fermentans
Binds to and Invades HeLa Cells: Involvement of Plasminogen and Urokinase
Authors: Yavlovich Amichai , Katzenell Avigail , Tarshis Mark , Higazi Abd A.-R. , Rottem Shlomo
Published: August 2004

Adherence of
Mycoplasma fermentans
to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37°C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that
M. fermentans
utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to
M. fermentans
greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound
M. fermentans
was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by
M. fermentans
. The latter finding indicates that the invasiveness of
M. fermentans
does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with β-cyclodextrin and filipin, respectively, did not affect the capacity of
M. fermentans
to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound
M. fermentans
was impaired, suggesting that lipid rafts are implicated in
M. fermentans
entry.
Microarray Analysis Reveals Induction of Lipoprotein Genes in MucoidPseudomonas aeruginosa: Implications for Inflammation in Cystic Fibrosis
Authors: Firoved Aaron M. , Ornatowski Wojciech , Deretic Vojo
Published: August 2004

The main cause of the high morbidity and mortality of cystic fibrosis (CF) is the progressive lung inflammation associated withPseudomonas aeruginosacolonization. During the course of chronic CF infections,P. aeruginosaundergoes a conversion to a mucoid phenotype. The emergence of mucoidP. aeruginosain CF is associated with increased inflammation, respiratory decline, and a poor prognosis. Here we show, by the use of microarray analysis, that uponP. aeruginosaconversion to mucoidy, there is a massive and preferential induction of genes encoding bacterial lipoproteins. Bacterial lipoproteins are potent agonists of Toll-like receptor 2 (TLR2) signaling. The expression of TLR2 in human respiratory epithelial cells was ascertained by Western blot analysis. Human respiratory epithelial cells responded in a TLR2-dependent manner to bacterial lipopeptides derived fromPseudomonaslipoproteins induced in mucoid strains. The TLR2 proinflammatory response was further augmented in CF cells. Thus, the excessive inflammation in CF is the result of a global induction in mucoidP. aeruginosaof lipoproteins that act as proinflammatory toxins (here termed lipotoxins) superimposed on the hyperexcitability of CF cells. Blocking the signaling cascade responding to bacterial lipotoxins may provide therapeutic benefits for CF patients.
Extracellular Signal-Regulated Protein Kinase Mediates Interleukin 17 (IL-17)-Induced IL-8 Secretion inHelicobacter pylori-Infected Human Gastric Epithelial Cells
Authors: Sebkova Ladislava , Pellicanò Antonia , Monteleone Giovanni , Grazioli Barbara , Guarnieri Giovanni , Imeneo Maria , Pallone Francesco , Luzza Francesco
Published: August 2004

Helicobacter pylori-induced mucosal inflammation results in high production of interleukin 17 (IL-17), a potent inducer of IL-8 in gastric epithelial cells. The aim of this study was to investigate signaling pathways by which IL-17 regulates IL-8 production in human gastric epithelial cells. Activation of mitogen-activated protein (MAP) kinases in both IL-17-stimulated MKN28 cells and epithelial cells isolated fromH. pylori-colonized gastric mucosa was assessed by Western blotting. In IL-17-stimulated MKN28 cells the activation of activatior protein 1 (AP-1), nuclear factor (NF)-IL-6, and NF-κB was also assessed by electrophoretic mobility shift assay. IL-8 production was evaluated by reverse transcription-PCR and enzyme-linked immunosorbent assay (ELISA) both for IL-17-stimulated MKN28 cells treated with specific MAP kinase inhibitors and gastric biopsy cultures treated with a neutralizing IL-17 antibody. Serum fromH. pylori-infected patients was tested for immunoglobulin G response to CagA by ELISA. Treatment of MKN28 cells with IL-17 caused activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) but not other MAP kinases and had the downstream effects of AP-1 and NF-κB activation and IL-8 synthesis. Blocking ERK 1/2 activity inhibited AP-1-mediated, but not NF-κB-mediated, IL-8 induction. Enhanced activation of ERK 1/2 was seen in gastric epithelial cells isolated fromH. pylori-infected patients in comparison to uninfected controls, and this was associated with high IL-8. These effects were even more pronounced in patients seropositive for CagA than in seronegative ones. In gastric biopsy cultures, the addition of a neutralizing IL-17 antibody decreased ERK 1/2 activation, thus resulting in a significant inhibition of IL-8. InH. pylori-colonized gastric epithelial cells, IL-17-induced IL-8 synthesis is associated with and depends at least in part on the activation of ERK 1/2 MAP kinase.
Intermittent Preventive Sulfadoxine-Pyrimethamine Treatment of Primigravidae Reduces Levels of Plasma Immunoglobulin G, Which Protects against Pregnancy-Associated
Plasmodium falciparum
Malaria
Authors: Staalsoe Trine , Shulman Caroline E , Dorman Edgar K. , Kawuondo Ken , Marsh Kevin , Hviid Lars
Published: August 2004

Pregnancy-associated malaria (PAM) is an important cause of maternal and neonatal suffering. It is caused by
Plasmodium falciparum
capable of inhabiting the placenta through expression of particular variant surface antigens (VSA) with affinity for proteoglycans such as chondroitin sulfate A. Protective immunity to PAM develops following exposure to parasites inhabiting the placenta, and primigravidae are therefore particularly susceptible to PAM. The adverse consequences of PAM in primigravidae are preventable by intermittent preventive treatment (IPTp), where women are given antimalarials at specified intervals during pregnancy, but this may interfere with acquisition of protective PAM immunity. We found that Kenyan primigravidae receiving sulfadoxine-pyrimethamine IPTp had significantly lower levels of immunoglobulin G (IgG) with specificity for the type of parasite-encoded VSA—called VSA
PAM
—that specifically mediate protection against PAM than did women receiving a placebo. VSA
PAM
-specific IgG levels depended on the number of IPTp doses received and were sufficiently low to be of clinical concern among multidose recipients. Our data suggest that IPTp should be extended to women of all parities, in line with current World Health Organization recommendations.
PspA Protects
Streptococcus pneumoniae
from Killing by Apolactoferrin, and Antibody to PspA Enhances Killing of Pneumococci by Apolactoferrin
Authors: Shaper Mirza , Hollingshead Susan K. , Benjamin William H. , Briles David E.
Published: August 2004

Lactoferrin is an important component of innate immunity through its sequestration of iron, bactericidal activity, and immune modulatory activity. Apolactoferrin (ALF) is the iron-depleted form of lactoferrin and is bactericidal against pneumococci and several other species of bacteria. We observed that lactoferricin (LFN), an 11-amino-acid peptide from the N terminus of lactoferrin, is bactericidal for
Streptococcus pneumoniae
. Strains of
S. pneumoniae
varied in their susceptibility to ALF. Lactoferrin is bound to the pneumococcal surface by pneumococcal surface protein A (PspA). Using mutant PspA
−
pneumococci of four different strains, we observed that PspA offers significant protection against killing by ALF. Knockout mutations in genes for two other choline-binding proteins (PspC and PcpA) did not affect killing by ALF. PspA did not have to be attached to the bacterial surface to inhibit killing, because the soluble recombinant N-terminal half of PspA could prevent killing by both ALF and LFN. An 11-amino-acid fragment of PspA was also able to reduce the killing by LFN. Antibody to PspA enhanced killing by lactoferrin. These findings suggested that the binding of ALF to PspA probably blocks the active site(s) of ALF that is responsible for killing.