bluuebunny/crossref_metadata_2025 huggingface dataset:
A Qualitative Study of Marital Process and Depressionin Older Couples*
Authors: Sandberg Jonathan G. , Miller Richard B. , Harper James M.
Published: February 2004

Although research has established that depression in later life can negatively impact the quality of an individual's life, few studies have addressed the relationship between depression and marital process in aging couples. To understand how older couples' experience and describe depression and its impact on their marriage, 26 older couples were interviewed. Results from 10 couples where one partner was experiencing depression and 16 nondepressed couples revealed that the depressed couples reported greater difficulty communicating and problem solving during “down” times. The nondepressed spouse in these couples reported feelings of confusion and frustration, whereas the depressed partner felt isolated and misunderstood. Implications for practice and future research are discussed.
Shelter in a Time of Storm: Parenting in Poor Rural African American Communities*
Authors: Wiley Angela R. , Warren Henriette Buur , Montanelli Dale S.
Published: February 2004

This study focuses on strengths and challenges of poor rural African American parents. Ninety‐two participants responded to semistructured interview questions about parenting, religiosity, and stress. Parents who are more religious reported using fewer coercive parenting strategies and experiencing fewer stressful life events. These findings are discussed in terms of practical implications for professionals who support rural African American families.
How Caregiving Grandparents View Support Groups for Grandchildren in Their Care*
Authors: Smith Gregory C. , Savage‐Stevens Susan E. , Fabian Ellen S.
Published: February 2004

Forty‐two grandparents who were primary caregivers to grandchildren completed self‐administered questionnaires and 28 also participated in focus groups to provide their views on the appropriateness and implementation of grandchildren support groups. The vast majority (88%) felt that such support groups would be beneficial, and few differences emerged regarding how they should be ideally implemented when comparing the views of grandparents varying in educational level, age of grandchildren, and status as a human service professional.
Rethinking Family Development Theory: Teaching With the Systemic Family Development (SFD) Model
Authors: Laszloffy Tracey A.
Published: February 2004

Although family development theory has made significant contributions to the field of family studies, virtually all of the models based on this theory are fraught with two fundamental conceptual weaknesses: the assumption of universality and the skew toward a single generational focus. The Systemic Family Developmental (SFD) Model is presented here as a process‐oriented and holistic alternative to existing models of family developmental theory. Following the presentation of a case example that demonstrates how the SFD Model can be used to study an actual family, detailed recommendations are provided for the use of the Model in undergraduate family development and family studies courses.
Did President Bush Mislead the Country in His Arguments for War with Iraq?
Authors: Pfiffner James P.
Published: February 2004

President Bush has been accused by some in the popular press of lying in his arguments for taking the United States to war with Iraq in 2003. This article examines several sets of statements by President Bush and his administration: first, about the implication that there was a link between Saddam Hussein, al Qaeda, and the terrorist attacks of 9/11; second, about Iraq's nuclear weapons capacity; and third, about Saddam Hussein's chemical and biological weapons and his ability to deliver them. Although the record at this early date is far from complete, the article concludes that from publicly available evidence, the president misled the country in important ways, potentially undermining the trust of the citizenry.
McKinley and the Spanish‐American War
Authors: Offner John L.
Published: February 2004

This article describes and analyzes President William McKinley's foreign and domestic policies that led to the Spanish‐American War of 1898. On the domestic side it includes congressional partisan politics, economic and business concerns, religious and moral views, cultural biases, and unexpected events that inflamed American patriotism. In foreign affairs it covers U.S. interests in Cuba, McKinley's diplomatic efforts to get Spain to withdraw peacefully from Cuba, and the president's relations with Europe's Great Powers and the pope. The article concludes with an analysis of McKinley's successes and failures.
Woodrow Wilson and World War I
Authors: Clements Kendrick A.
Published: February 2004

The United States declared war on Germany and entered World War I on April 6, 1917, but the crucial decision that led inexorably to that moment had been made two years earlier. In demanding that German submarines obey traditional rules of surface warfare, the Wilson administration turned over to the Germans the choice between war and peace. That policy was less the result of rational analysis of a particular challenge to American interests than the outcome of complex interactions among assumptions and personalities in the months between August 1914 and February 1915.
Franklin D. Roosevelt and World War II
Authors: Kimball Warren F.
Published: February 2004

“Roosevelt ‘had said that he would wage war, but not declare it, and that he would become more and more provocative.’ ”
Lyndon Johnson and Vietnam
Authors: Logevall Fredrik
Published: February 2004

Lyndon Johnson's decision to take the United States into large‐scale war in Vietnam in 1964‐1965 must loom large in any evaluation of his presidency. This article argues that the decision was not forced on Johnson by pressures beyond his control, and that his choice for war is difficult to explain, in view of the international and domestic political context, the dynamics on the ground in Vietnam, and the thinking among U.S. officials themselves. The article maintains that Johnson could expect—on the basis of information available to him at the time—to pay a heavy political price if he Americanized the war, and examines why he nevertheless did so.
George Bush and the Gulf War of 1991
Authors: Brands H. W.
Published: February 2004

On August 2, 1990, Iraqi troops invaded Kuwait. Within 24 hours they controlled that small country. On January 16, 1991, President George Bush ordered U.S. military forces into battle to undo the Iraqi conquest and liberate Kuwait. The president's decision for war came in several stages. In the first stage, which lasted hardly longer than it took for Saddam Hussein's troops to subdue Kuwait, Bush determined that Iraqi soldiers must be expelled from Kuwait and that country's sovereignty restored. In announcing this decision on the third day after the invasion, he did not say explicitly that force might be used to accomplish this goal, but the implication was obvious and deliberate. In the second stage of the decision process, which lasted from early August until late October, Bush deployed American forces to Saudi Arabia and orchestrated sanctions against Iraq. The deployment of forces, initially designed to defend Saudi Arabia against further Iraqi adventurism, increasingly afforded the Bush administration the capacity to wage war against Iraq; this capacity, together with the sanctions, was intended to signal the seriousness of the United States and its allies. The role of allies became more explicit during the third stage of the decision process, in which the administration pressed for, and in late November received, explicit authorization from the United Nations Security Council for the use of force to evict Iraq from Kuwait. In the final stage of the decision process, Bush focused on the home front, seeking—and, again, finally receiving—congressional endorsement of war against Iraq. Days after the vote, the president ordered American forces, in conjunction with those of several allies, to war.
The Contemporary Presidency: George W. Bush and the Myth of Heroic Presidential Leadership
Authors: Roper Jon
Published: February 2004

This article discusses the reinvention of George W. Bush's image during a critical period of his presidency—the 18 months after September 11, 2001. It examines how, during that time, he assumed a mantle of heroic presidential leadership similar to that designed by a previous generation for one of his predecessors: John F. Kennedy. In the “age of infotainment,” however, it remains a challenge for presidents to make the transition from “celebrity” to “hero.” While the currency of celebrity is easy to acquire, the political capital that accrues to the heroic leader in times of national crisis is more difficult to keep. In pursuing the “war on terror,” George W. Bush must beware the “credibility gap” that can impact on the presidency if a president picks the wrong fight and loses control of the way his image is mediated.
Elections: Personal Popularity in U.S. Presidential Elections
Authors: Wattenberg Martin P.
Published: February 2004

A commonly held view is that presidential elections are largely personality contests, and that the candidate with the best‐liked personality wins. But is this really the case? Based on a careful analysis of national survey data from the last 11 presidential elections, this article concludes that such a view is unfounded. The most personally popular candidate does not always win in the U.S.—indeed, in recent elections, the most personally popular candidate has generally lost. Much more central to candidate evaluations, and to who emerges victorious, are perceptions of candidates’ stands on the issues.
The Law: Termination of the ABM Treaty and the Political Question Doctrine: Judicial Succor for Presidential Power
Authors: Adler David Gray
Published: February 2004

President George W. Bush's unilateral termination of the 1972 ABM Treaty between the United States and Russia triggered a lawsuit, Kucinich v. Bush, in which members of the House of Representatives challenged the constitutionality of Bush's action on grounds that the president may not terminate a treaty without congressional approval. The Federal District Court refused to reach the merits of the case and dismissed the case as a nonjusticiable political question. The court's action reflects a disturbing tendency among lower court judges to elide substantive issues in foreign relations cases that challenge presidential usurpation of power. As a result, presidential aggrandizement of power remains unchecked.
Role of MyD88 in Diminished Tumor Necrosis Factor Alpha Production by Newborn Mononuclear Cells in Response to Lipopolysaccharide
Authors: Yan Sen Rong , Qing Gefei , Byers David M. , Stadnyk Andrew W. , Al-Hertani Walla , Bortolussi Robert
Published: February 2004

Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-α) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-α. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-α compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-α secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IκBα phosphorylation, as well as IκBα degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-α secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-α secretion in newborn monocytes.
Contribution of Long Polar Fimbriae to the Virulence of Rabbit-Specific Enteropathogenic
Escherichia coli
Authors: Newton Hayley J. , Sloan Joan , Bennett-Wood Vicki , Adams Louise M. , Robins-Browne Roy M. , Hartland Elizabeth L.
Published: February 2004

Enteropathogenic
Escherichia coli
(EPEC) is a major of cause of diarrhea among children in developing countries. Although EPEC is a human specific pathogen, some related strains are natural pathogens of animals, including laboratory-bred rabbits. We have identified two chromosomal loci in rabbit-specific EPEC (REPEC) O15:H− strain 83/39, which are predicted to encode long polar fimbriae (LPF).

lpf
R154

was identical to a fimbrial gene cluster,

lpf
O113

, identified previously in enterohemorrhagic
E. coli
(EHEC) O113:H21. The second locus,

lpf
R141

, comprised a novel sequence with five predicted open reading frames,
lpfA
to
lpfE
, that encoded long fine fimbriae in nonfimbriated
E. coli
ORN103. The predicted products of

lpf
R141

shared identity with components of the
lpfABCC′DE
gene cluster from EHEC O157:H7, and the fimbriae were similar in morphology and length to LPF from EHEC O157:H7. Interruption of

lpf
R141

resulted in significant attenuation of REPEC 83/39 for rabbits with respect to the early stages of colonization and severity of diarrhea. However, there was no significant difference in the number of bacteria shed at later time points or in overall body weight and mortality rate of rabbits infected with

lpf
R141

mutant strains or wild-type REPEC 83/39. Although rabbits infected with the

lpf
R141

mutants did not develop severe diarrhea, there was evidence of attaching and effacing histopathology, which was indistinguishable in morphology, location, and extent compared to rabbits infected with wild-type REPEC 83/39. The results suggested that

lpf
R141

contributes to the early stages of REPEC-mediated disease and that this is important for the development of severe diarrhea in susceptible animals.
A Type III Secretion System Is Required for
Aeromonas hydrophila
AH-1 Pathogenesis
Authors: Yu H. B. , Rao P. S. Srinivasa , Lee H. C. , Vilches S. , Merino S. , Tomas J. M. , Leung K. Y.
Published: February 2004

Aeromonas hydrophila
is a gram-negative opportunistic pathogen in fish and humans. Many bacterial pathogens of animals and plants have been shown to inject anti-host virulence determinants into the hosts via a type III secretion system (TTSS). Degenerate primers based on
lcrD
family genes that are present in every known TTSS allowed us to locate the TTSS gene cluster in
A. hydrophila
AH-1. A series of genome walking steps helped in the identification of 25 open reading frames that encode proteins homologous to those in TTSSs in other bacteria. PCR-based analysis showed the presence of
lcrD
homologs (
ascV
) in all of the 33 strains of
A. hydrophila
isolated from various sources. Insertional inactivation of two of the TTSS genes (
aopB
and
aopD
) led to decreased cytotoxicity in carp epithelial cells, increased phagocytosis, and reduced virulence in blue gourami. These results show that a TTSS is required for
A. hydrophila
pathogenesis. This is the first report of sequencing and characterization of TTSS gene clusters from
A. hydrophila
. The TTSS identified here may help in developing suitable vaccines as well as in further understanding of the pathogenesis of
A. hydrophila
.
A 38-Kilobase Pathogenicity Island Specific forMycobacterium aviumsubsp.paratuberculosisEncodes Cell Surface Proteins Expressed in the Host
Authors: Stratmann Janin , Strommenger Birgit , Goethe Ralph , Dohmann Karen , Gerlach Gerald-F. , Stevenson Karen , Li Ling-ling , Zhang Qing , Kapur Vivek , Bull Tim J.
Published: February 2004

We have used representational difference analysis to identify a novelMycobacterium aviumsubsp.paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designatedmptAto -Fand is immediately preceded by two putative Fur boxes. Functional genomics revealed that themptoperon is flanked on one end by afepcluster encoding proteins involved in the uptake of Fe3+and on the other end by asidcluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kbM. aviumsubsp.paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction ofM. aviumsubsp.paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening ofMycobacterium smegmatistransformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes anM. aviumsubsp.paratuberculosispathogenicity island.
Purification and Analysis of a Phospholipase A2-Like Lytic Factor ofTrichomonas vaginalis
Authors: Lubick Kirk J. , Burgess Donald E.
Published: February 2004

Trichomonas vaginalisproduces soluble factors that have been reported to have the ability to damage target cells in vitro, and it has been hypothesized that these factors may play a role in the pathogenesis of human trichomoniasis. A lytic factor (LF) was purified fromT. vaginalis, and the molecular characteristics of LF were determined.T. vaginalisextract was subjected to hydrophobic chromatography with a 10 to 60%N-propanol gradient in 0.1 M ammonium acetate, resulting in the elution of LF from the column at 30%N-propanol. Cytotoxicity assays revealed that LF was cytotoxic to WEHI 164 cells and bovine red blood cells, and inactivation of LF by treatment with trypsin suggested that the active component of LF was a protein. Size exclusion chromatography of LF produced two fractions at 144 and 168 kDa, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LF under reducing conditions revealed two subunits of 57 and 60 kDa. Results of a fluorescence assay of LF on carboxyfluorescein-labeled liposomes composed of phosphatidylcholine-cholesterol showed that liposomes were hydrolyzed, suggesting that LF had phospholipase activity. Thin-layer chromatography analysis of BODIPY (4,4-difluoro-3a,4adiaza-s-indacene)-labeled phosphatidylcholine treated with LF demonstrated products that migrated identically to the products produced by treatment with phospholipase A2(PLA2). These results suggest that LF is a PLA2and may be an important virulence factor ofT. vaginalismediating the destruction of host cells and contributing to tissue damage and inflammation in trichomoniasis.
Lutzomyia longipalpisSalivary Gland Homogenate Impairs Cytokine Production and Costimulatory Molecule Expression on Human Monocytes and Dendritic Cells
Authors: Costa Dirceu J. , Favali Cecília , Clarêncio Jorge , Afonso Lílian , Conceição Viviane , Miranda José Carlos , Titus Richard G. , Valenzuela Jesus , Barral-Netto Manoel , Barral Aldina , Brodskyn Cláudia Ida
Published: February 2004

In this report, we describe an investigation of the effects ofLutzomyia longipalpissand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Mφs), and dendritic cells (DCs). SGH ofL. longipalpisinduced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Mφs, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Mφs, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SGH-specific antibodies abolished the effects of SGH on cytokine production by LPS-stimulated monocytes.
Cryptosporidium parvum-Specific CD4 Th1 Cells from Sensitized Donors Responding to Both Fractionated and Recombinant Antigenic Proteins
Authors: Morales Maria Angeles Gomez , Mele Raffaella , Ludovisi Alessandra , Bruschi Fabrizio , Tosini Fabio , Pozio Edoardo
Published: February 2004

T-cell-mediated immunity plays a central role in the host response toCryptosporidium parvum. Human T-cell clones (TCC) were isolated from peripheral blood mononuclear cells of five healthy donors with prior cryptosporidiosis by use of aC. parvumcrude extract, two antigen fractions obtained by ion-exchange chromatography (IEC1 and IEC2), and two recombinant peptides (SA35 and SA40) fromC. parvumsporozoites. The T-cell lines derived from the one recently infected donor had a higher proportion (26 to 38%) of T cells exhibiting the γ/δ T-cell receptor (γ/δ-TCR) than those from donors who had recovered from cryptosporidiosis several years earlier, suggesting that the γ/δ T-cell population is involved in the early stage of the infection. The specific TCC had the α/β-TCR, had the phenotype CD45RO+CD4+CD8−, and were characterized by either hyperproduction of gamma interferon (IFN-γ) alone, with a Th1 profile, or IFN-γ hyperproduction together with interleukin-4 (IL-4) or IL-5 production, with a Th0 profile. SA35, SA40, IEC1, and IEC2 may be considered good targets of the cellular response againstC. parvumand may play a role in maintaining the T-cell-mediated memory response to this parasite. Furthermore, the SA35 and SA40 peptides may be regarded as immunodominant antigens involved in the maintenance of the T-cell response in healthyC. parvum-sensitized persons.
Production of Interleukin-8 by Human Neutrophils Stimulated with
Trichomonas vaginalis
Authors: Ryu Jae-Sook , Kang Ji-Hyun , Jung Seung-Yong , Shin Myeong-Heon , Kim Jung-Mogg , Park Hyun , Min Duk-Young
Published: February 2004

Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients infected with
Trichomonas vaginalis
. Although chemoattractants, such as leukotriene B
4
and interleukin-8 (IL-8), are found in the vaginal discharges of symptomatic trichomoniasis patients, little is known about the mechanism of how neutrophils accumulate or mediate initial inflammatory response after acute
T. vaginalis
infection. We examined IL-8 production in neutrophils activated by
T. vaginalis
and evaluated the factors involved in
T. vaginalis
adherence that might affect IL-8 production. When human neutrophils were stimulated with live trophozoites,
T. vaginalis
lysate, or
T. vaginalis
excretory-secretory products, the live trichomonads induced higher levels of IL-8 production than the lysate or products did. When live trichomonads were pretreated with various inhibitors of proteinase, microtubule, microfilament, or adhesin (which are all known to participate in the adherence of
T. vaginalis
to vaginal epithelial cells), IL-8 production significantly decreased compared with the untreated controls. Furthermore, an NF-κB inhibitor (pyrrolidine dithiocarbamate), a mitogen-activated protein (MAP) kinase (MEK) inhibitor (PD98059), and a p38 MAP kinase inhibitor (SB203580) significantly suppressed IL-8 synthesis in neutrophils. These results suggest that live
T. vaginalis
, particularly adherent trophozoites, can induce IL-8 production in neutrophils and that this action may be mediated through the NF-κB and MAP kinase signaling pathways. In other words,
T. vaginalis
-induced neutrophil recruitment may be mediated via the IL-8 expressed by neutrophils in response to activation by live
T. vaginalis
.
Identification of Novel Virulence-Associated Genes via Genome Analysis of Hypothetical Genes
Authors: Garbom Sara , Forsberg Åke , Wolf-Watz Hans , Kihlberg Britt-Marie
Published: February 2004

The sequencing of bacterial genomes has opened new perspectives for identification of targets for treatment of infectious diseases. We have identified a set of novel virulence-associated genes (
vag
genes) by comparing the genome sequences of six human pathogens that are known to cause persistent or chronic infections in humans:
Yersinia pestis
,
Neisseria gonorrhoeae
,
Helicobacter pylori
,
Borrelia burgdorferi
,
Streptococcus pneumoniae
, and
Treponema pallidum.
This comparison was limited to genes annotated as hypothetical in the
T. pallidum
genome project. Seventeen genes with unknown functions were found to be conserved among these pathogens. Insertional inactivation of 14 of these genes generated nine mutants that were attenuated for virulence in a mouse infection model. Out of these nine genes, five were found to be specifically associated with virulence in mice as demonstrated by infection with
Yersinia pseudotuberculosis
in-frame deletion mutants. In addition, these five
vag
genes were essential only in vivo, since all the mutants were able to grow in vitro. These genes are broadly conserved among bacteria. Therefore, we propose that the corresponding
vag
gene products may constitute novel targets for antimicrobial therapy and that some
vag
mutants could serve as carrier strains for live vaccines.
Regulation of Adenosine Receptor Subtypes during Cultivation of Human Monocytes: Role of Receptors in Preventing Lipopolysaccharide-Triggered Respiratory Burst
Authors: Thiele Andrea , Kronstein Romy , Wetzel Anne , Gerth Anja , Nieber Karen , Hauschildt Sunna
Published: February 2004

Adenosine is a potent anti-inflammatory agent that modulates the function of cells involved in the inflammatory response. Here we show that it inhibits lipopolysaccharide (LPS)-induced formation of reactive oxygen intermediates (ROI) in both freshly isolated and cultured human monocytes. Blocking of adenosine uptake and inactivation of the adenosine-degrading enzyme adenosine deaminase enhanced the inhibitory action of adenosine, indicating that both pathways regulate the extracellular adenosine concentration. Adenosine-mediated inhibition could be reversed by XAC (xanthine amine congener), an antagonist of the adenosine receptor A2A, and MRS 1220 {N-9-chloro-2-(2-furanyl)[1, 2, 4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide}, an A3receptor antagonist, in both cell populations, while DPCPX (1,3-dipropyl-8-cyclopentylxanthine), an A1receptor antagonist, had no effect. Similar to what was seen with adenosine, CGS 21680, an A2Aand A3receptor agonist, and IB-MECA, a nonselective A1and A3receptor agonist, dose dependently prevented ROI formation, indicating the involvement of A3and probably also A2Ain the suppressive effect of adenosine. Pretreatment of monocytes with adenosine did not lead to changes in the LPS-induced increase in intracellular calcium levels ([Ca2+]i). Thus, participation of [Ca2+]iin the action of adenosine seems unlikely. The adenosine-mediated suppression of ROI production was found to be more pronounced when monocytes were cultured for 18 h, a time point at which changes in the mRNA expression of adenosine receptors were observed. Most prominent was the increase in the A2Areceptor mRNA. These data demonstrate that cultivation of monocytes is accompanied by changes in the inhibitory action of adenosine mediated by A3and probably also the A2Areceptor and that regulation of adenosine receptors is an integral part of the monocyte differentiation program.
The DnaK/DnaJ Chaperone Machinery of
Salmonella enterica
Serovar Typhimurium Is Essential for Invasion of Epithelial Cells and Survival within Macrophages, Leading to Systemic Infection
Authors: Takaya Akiko , Tomoyasu Toshifumi , Matsui Hidenori , Yamamoto Tomoko
Published: February 2004

Salmonella enterica
serovar Typhimurium, similar to various facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophages of the host organism by inducing stress proteins, such as DnaK. DnaK forms a chaperone machinery with the cochaperones DnaJ and GrpE. To elucidate the role of the DnaK chaperone machinery in the pathogenesis of
S. enterica
serovar Typhimurium, we first constructed an insertional mutation in the
dnaK
-
dnaJ
operon of pathogenic strain χ3306. The DnaK/DnaJ-depleted mutant was temperature sensitive for growth, that is, nonviable above 39°C. We then isolated a spontaneously occurring revertant of the
dnaK
-
dnaJ
-disrupted mutant at 39°C and used it for infection of mice. The mutant lost the ability to cause a lethal systemic disease in mice. The impaired ability for virulence was restored when a functional copy of the
dnaK
-
dnaJ
operon was provided, suggesting that the DnaK/DnaJ chaperone machinery is required by
Salmonella
for the systemic infection of mice. This result also indicates that with respect to the DnaK/DnaJ chaperone machinery, the cellular requirements for growth at a high temperature are not identical to the cellular requirements for the pathogenesis of
Salmonella
. Macrophage survival assays revealed that the DnaK/DnaJ-depleted mutant could not survive or proliferate at all within macrophages. Of further interest are the findings that the mutant could neither invade cultured epithelial cells nor secrete any of the invasion proteins encoded within
Salmonella
pathogenicity island 1. This is the first time that the DnaK/DnaJ chaperone machinery has been shown to be involved in bacterial invasion of epithelial cells.
Effect of Metabolic Imbalance on Expression of Type III Secretion Genes in
Pseudomonas aeruginosa
Authors: Rietsch Arne , Wolfgang Matthew C. , Mekalanos John J.
Published: February 2004

The type III secretion system is a dedicated machinery used by many pathogens to deliver toxins directly into the cytoplasm of a target cell. Expression and secretion of the type III effectors are triggered by cell contact. In
Pseudomonas aeruginosa
and
Yersinia
spp., expression can be triggered in vitro by removing calcium from the medium. The mechanism underlying either mode of regulation is unclear. Here we characterize a transposon insertion mutant of
P. aeruginosa
PAO1 that displays a marked defect in cytotoxicity. The insertion is located upstream of several genes involved in histidine utilization and impedes the ability of PAO1 to intoxicate eukaryotic cells effectively in a type III-dependent fashion. This inhibition depends on the presence of histidine in the medium and appears to depend on the excessive uptake and catabolism of histidine. The defect in cytotoxicity is mirrored by a decrease in
exoS
expression. Other parameters such as growth or piliation are unaffected. The cytotoxicity defect is partially complemented by an insertion mutation in
cbrA
that also causes overexpression of
cbrB
. The
cbrAB
two-component system has been implicated in sensing and responding to a carbon-nitrogen imbalance. Taken together, these results suggest that the metabolic state of the cell influences expression of the type III regulon.
An NADPH Quinone Reductase of
Helicobacter pylori
Plays an Important Role in Oxidative Stress Resistance and Host Colonization
Authors: Wang Ge , Maier Robert J.
Published: February 2004

Oxidative stress resistance is one of the key properties that enable pathogenic bacteria to survive the toxic reactive oxygen species released by the host. In a previous study characterizing oxidative stress resistance mutants of
Helicobacter pylori
, a novel potential antioxidant protein (MdaB) was identified by the observation that the expression of this protein was significantly upregulated to compensate for the loss of other major antioxidant components. In this study, we characterized an
H. pylori mdaB
mutant and the MdaB protein. While the wild-type strain can tolerate 10% oxygen for growth, the growth of the
mdaB
mutant was significantly inhibited by this oxygen condition. The
mdaB
mutant is also more sensitive to H
2
O
2
, organic hydroperoxides, and the superoxide-generating agent paraquat. Although the wild-type strain can survive more than 10 h of air exposure, exposure of the mutant strain to air for 8 h resulted in recovery of no viable cells. The oxidative stress sensitivity of the
mdaB
mutant resulted in a deficiency in the ability to colonize mouse stomachs.
H. pylori
was recovered from 10 of 11 mouse stomachs inoculated with the wild-type strain, with about 5,000 to 45,000 CFU/g of stomach. However, only 3 of 12 mice that were inoculated with the
mdaB
mutant strain were found to harbor any
H. pylori
, and these 3 contained less than 2,000 CFU/g of stomach. A His-tagged MdaB protein was purified and characterized. It was shown to be a flavoprotein that catalyzes two-electron transfer from NAD(P)H to quinones. It reduces both ubiquinones and menaquinones with similar efficiencies and preferably uses NADPH as an electron donor. We propose that the physiological function of the
H. pylori
MdaB protein is that of an NADPH quinone reductase that plays an important role in managing oxidative stress and contributes to successful colonization of the host.
Effects of RelA on Key Virulence Properties of Planktonic and Biofilm Populations ofStreptococcus mutans
Authors: Lemos José A. C. , Brown Thomas A. , Burne Robert A.
Published: February 2004

Streptococcus mutansis a biofilm-forming bacterium that is adapted to tolerate rapid and dramatic fluctuations in nutrient availability, carbohydrate source, and pH in its natural environment, the human oral cavity. Dissecting the pathways used to form stable biofilms and to tolerate environmental stress is central to understanding the virulence of this organism. Here, we investigated the role of theS. mutans relAgene, which codes for a guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase/hydrolase, in biofilm formation and acid tolerance. Two mutants in whichrelAwas insertionally inactivated or replaced by an antibiotic resistance determinant were constructed. Under normal growth and stress conditions, the mutants grew slower than the wild-type strain, although the final yields were similar. The mutants, which were still able to accumulate (p)ppGpp after the induction of a stringent response, showed significant reductions in biofilm formation on microtiter plates or hydroxylapatite disks. There was no difference in the sensitivities to acid killing of the parent andrelAstrains grown in planktonic cultures. However, when cells were grown in biofilms, the mutants became more acid resistant and could lower the pH through glycolysis faster and to a greater extent than the wild-type strain. Differences in acid resistance were not correlated with increases in F-ATPase activity, although bacterial sugar:phosphotransferase activity was elevated in the mutants. Expression of theluxSgene was increased as much as fivefold in therelAmutants, suggesting a link between AI-2 quorum sensing and the stringent response.
Invasion of Porcine Brain Microvascular Endothelial Cells by
Streptococcus suis
Serotype 2
Authors: Vanier Ghyslaine , Segura Mariela , Friedl Peter , Lacouture Sonia , Gottschalk Marcelo
Published: February 2004

Streptococcus suis
is an important swine pathogen that mainly causes meningitis and occasionally causes other infections, such as endocarditis, arthritis, and pneumonia. The pathogenesis of
S. suis
infection has not been completely defined. However, in order to cause meningitis,
S. suis
has to cross the blood-brain barrier (BBB) made up of brain microvascular endothelial cells. The objective of this work was to study the interactions of
S. suis
serotype 2 with porcine brain microvascular endothelial cells (PBMEC). The ability of North American and European
S. suis
serotype 2 strains to adhere to PBMEC and, most importantly, to invade PBMEC was demonstrated by using an antibiotic protection assay and was confirmed by electron microscopy. The polysaccharide capsule of
S. suis
seemed to partially interfere with the adhesion and invasion abilities of the bacterium. Our results showed that intracellular viable
S. suis
could be found in PBMEC up to 7 h after antibiotic treatment. Inhibition studies demonstrated that invasion of PBMEC by
S. suis
required actin microfilaments but not microtubular cytoskeletal elements or active bacterial RNA or protein synthesis. At high bacterial doses, suilysin-positive strains were toxic for PBMEC. The role of suilysin in cytotoxicity was confirmed by using purified suilysin, electron microscopy, and the lack of toxicity of a suilysin-negative mutant. In swine, the invasion of endothelial cells of the BBB could play an important role in the pathogenesis of the meningitis caused by
S. suis
.
Paneth Cell α-Defensins from Rhesus Macaque Small Intestine
Authors: Tanabe Hiroki , Yuan Jun , Zaragoza Melinda M. , Dandekar Satya , Henschen-Edman Agnes , Selsted Michael E. , Ouellette Andre J.
Published: February 2004

Antimicrobial peptides are secreted by small intestinal Paneth cells as components of innate immunity. To investigate the role of α-defensins in enteric host defenses in nonhuman primates, α-defensin cDNAs were isolated, α-defensin peptides were purified from rhesus macaque small bowel, and the bactericidal activities of the peptides were measured. Six rhesus enteric α-defensin (RED) cDNAs, RED-1 to RED-6, were identified in a jejunum cDNA library; the deduced RED peptides exhibited extensive diversity relative to the primary structures of rhesus myeloid α-defensins. RED-4 was purified from monkey jejunum, and N-terminal peptide sequencing of putative RED-4 peptides identified two N termini, RTCYCRTGR… and TCYCRTGRC…; these corresponded to alternative N termini for the RED-4 molecules, as deduced from their molecular masses and RED cDNAs. In situ hybridization experiments localized RED mRNAs exclusively to small intestinal Paneth cells. Recombinant RED-1 to RED-4 were purified to homogeneity and shown to be microbicidal in the low micromolar range (≤10 μg/ml) against gram-positive and gram-negative bacteria, with individual peptides exhibiting variable target cell specificities. Thus, compared to myeloid α-defensins from rhesus macaques, enteric α-defensin peptides are highly variable in both primary structure and activity. These studies should facilitate further analyses of the role of α-defensins in primate enteric immunity.
γδ T Cells in Immunity Induced byMycobacterium bovisBacillus Calmette-Guérin Vaccination
Authors: Lee Jinhee , Choi Keumhwa , Olin Michael R. , Cho Sang-Nae , Molitor Thomas W.
Published: February 2004

Mycobacterium bovisbacillus Calmette-Guérin (BCG) vaccination is efficacious for newborns or adults with no previous exposure to environmental mycobacteria. To determine the relative contribution and the nature of γδ T-cell receptor-positive T cells in newborns, compared to CD4+T cells, in immunity induced byM. bovisBCG vaccination, 4-week-old specific-pathogen-free pigs were vaccinated withM. bovisBCG and monitored by following the γδ T-cell immune responses. A flow cytometry-based proliferation assay and intracellular staining for gamma interferon (IFN-γ) were used to examine γδ T-cell responses. Pigs were found to mount Th1-like responses toM. bovisBCG vaccination as determined by immunoproliferation and IFN-γ production. The γδ T-cell lymphoproliferation and IFN-γ production to stimulation with mycobacterial antigens were significantly enhanced byM. bovisBCG vaccination. The relative number of proliferating γδ T cells after stimulating peripheral blood mononuclear cells withMycobacterium tuberculosisH37Rv culture filtrate protein was higher than that of CD4+T cells at an early time point afterM. bovisBCG vaccination, but CD4+T cells were found to be more abundant at a later time point. Although the γδ T-cell responses were dependent on the presence of CD4+T cells for the cytokine interleukin-2, the enhanced γδ T cells were due to the intrinsic changes of γδ T cells caused byM. bovisBCG vaccination rather than being due solely to help from CD4+T cells. Our study shows that γδ T cells from pigs at early ages are functionally enhanced byM. bovisBCG vaccination and suggests an important role for this T-cell subset in acquired immunity conferred byM. bovisBCG vaccination.
Increases in c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase and p38 Activity in Monocyte-Derived Macrophages following the Uptake of
Legionella pneumophila
Authors: Welsh Chad T. , Summersgill James T. , Miller Richard D.
Published: February 2004

Legionella pneumophila
, the causative agent of Legionnaires' disease, infects and replicates within a variety of eukaryotic cells. The purpose of the current study was to examine host cell signaling events immediately following uptake and early in the endocytic process (less than 1 h) following the phagocytosis of
L. pneumophila
. This examination focused on the protein kinase signal pathways to identify any aberrant signal(s) induced by
L. pneumophila
within its host, as a means to alter the normal endocytic pathway. The mitogen-activated protein kinase cascades are of interest due to their involvement in cellular regulation. The experiments were carried out with monocyte-derived macrophages (MDMs). All three mitogen-activated protein kinase cascades were activated when MDMs were inoculated with either
Legionella
strain (wild-type strain AA100 or
dotA
mutant GL10) or an
Escherichia coli
control. Whereas the avirulent treatments, GL10 and
E. coli
, exhibited a leveling off or a return to near basal levels of phosphorylation/activity of c-Jun N-terminal kinase by 60 min, the virulent strain AA100 exhibited a significantly increased level of activity through 60 min that was greater than that seen in GL10 (
P
= 0.025) and
E. coli
(
P
= 0.014). A similar trend was seen with p38 phosphorylation. Phosphorylation of mitogen-activated protein/ERK kinase (MEK) was decreased in strain AA100 compared to
E. coli
. Inhibition of the activity of either the stress-activated protein kinase/c-Jun N-terminal kinase or p38 pathway significantly decreased the ability of legionellae to replicate intracellularly, suggesting the necessity of these two pathways in its intracellular survival and replication.
Populations of Human T Lymphocytes That Traverse the Vascular Endothelium Stimulated byBorrelia burgdorferiAre Enriched with Cells That Secrete Gamma Interferon
Authors: Gergel Edna I. , Furie Martha B.
Published: February 2004

Some diseases are characterized by prevalence in the affected tissues of type 1 T lymphocytes, which secrete gamma interferon (IFN-γ) and other proinflammatory cytokines. For example, type 1 T cells predominate in the lesions of patients with Lyme disease, which is caused by the bacteriumBorrelia burgdorferi. We used an in vitro model of the blood vessel wall to test the premise that the vascular endothelium actively recruits circulating type 1 T cells to such lesions. When T lymphocytes isolated from human peripheral blood were examined, the populations that traversed monolayers of resting human umbilical vein endothelial cells (HUVEC) or HUVEC stimulated by interleukin-1β orB. burgdorferiwere markedly enriched for T cells that produced IFN-γ compared to the initially added population of T cells. No enrichment was seen for cells that produced interleukin-4, a marker for type 2 T lymphocytes. Very late antigen-4 and CD11/CD18 integrins mediated passage of the T cells across both resting and stimulated HUVEC, and the endothelium-derived chemokine CCL2 (monocyte chemoattractant protein 1) was responsible for the enhanced migration of T cells across stimulated HUVEC. These results suggest that the vascular endothelium may contribute to the selective accumulation of type 1 T cells in certain pathological lesions, including those of Lyme disease.
TheBurkholderia cepaciaEpidemic Strain Marker Is Part of a Novel Genomic Island Encoding Both Virulence and Metabolism-Associated Genes inBurkholderia cenocepacia
Authors: Baldwin Adam , Sokol Pamela A. , Parkhill Julian , Mahenthiralingam Eshwar
Published: February 2004

TheBurkholderia cepaciaepidemic strain marker (BCESM) is a useful epidemiological marker for virulentB. cenocepaciastrains that infect patients with cystic fibrosis. However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity. Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism. The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): anN-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes. Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out. Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence. The island, designated theB. cenocepaciaisland (cci), is the first genomic island to be defined in theB. cepaciacomplex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains. The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.
Epitope Mapping of a Protective Monoclonal Antibody against
Pneumocystis carinii
with Shared Reactivity to
Streptococcus pneumoniae
Surface Antigen PspA
Authors: Wells Jesse , Gigliotti Francis , Simpson-Haidaris Patricia J. , Haidaris Constantine G.
Published: February 2004

Pneumocystis carinii
is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived
P. carinii
was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of
P. carinii
derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse
P. carinii
antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse
P. carinii
recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen,
Streptococcus pneumoniae
PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the
S. pneumoniae
surface, and Western blotting identified a protein in
S. pneumoniae
lysates consistent with the

M
r

of PspA. A fragment of the
S. pneumoniae
PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in
P. carinii
. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize
P. carinii
derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against
P. carinii
.