bluuebunny/crossref_metadata_2025 huggingface dataset:
Schirmer tear test values and tear film break‐up times in cats with conjunctivitis
Authors: Lim Christine C. , Cullen Cheryl L.
Published: September 2005

Objectives (1) To document tear film break‐up time (TFBUT) in a group of cats with conjunctivitis; (2) to determine if TFBUTs from cats with conjunctivitis vary significantly from previously established normal values for TFBUT in young cats without ocular disease; (3) to determine if a correlation exists between Schirmer tear test (STT) values and TFBUTs in cats with conjunctivitis; (4) to determine if the TFBUTs in cats with conjunctivitis are influenced by the detection of DNA from feline herpes virus‐type 1 (FHV‐1),Chlamydophila felis,Mycoplasmaspp., and feline calicivirus.

Animals studied Fourteen cats between the ages of 0.8 years to 12 years with active, untreated conjunctivitis and without active keratitis or other ocular or systemic abnormalities were included in this study.

Procedures Complete ophthalmic examinations, including TFBUT, were performed on all cats. Polymerase chain reaction (PCR) screening for FHV‐1,Chlamydophila felis,Mycoplasmaspp., and feline calicivirus was performed on conjunctival swabs from affected eyes and blood samples from all cats.

Results Mean TFBUT for cats in this study was 8.9 (± 4.8) s in the right eye (OD) and 8.1 (± 4.6) s in the left eye (OS). No correlation existed between mean TFBUTs and mean STT values OD or OS. Conjunctival swabs from seven cats (n = 9 eyes) tested positive via PCR for one of the above infectious agents. Blood samples from nine cats tested positive for FHV‐1. Mean TFBUTs for cats from which the DNA from FHV‐1 was isolated from the blood were significantly lower than mean TFBUTs for cats from which no such DNA was isolated from the blood.

Conclusions In this study, the mean TFBUT in cats with conjunctivitis was significantly lower than previously established values for clinically healthy cats. This supports the theory that qualitative tear film deficiency, and thus tear film instability, may play a role in the pathogenesis of feline conjunctivitis. Qualitative tear film deficiency may predispose to the development of conjunctivitis or may occur secondarily to this condition.
Oral doxycycline, niacinamide and prednisolone used to treat bilateral nodular granulomatous conjunctivitis of the third eyelid in an Australian Kelpie dog
Authors: Hurn Simon , Mc Cowan Christina , Turner Andrew
Published: September 2005

A 5‐year‐old, female neutered, Australian Kelpie presented with a 2‐month history of dramatic bilateral erythematous thickening of the third eyelids. Ophthalmic examination demonstrated raised, pink to red, irregular thickening of the entire palpebral surface of both third eyelids. There were no other ocular abnormalities. A surgical biopsy was taken from each third eyelid. Histopathologic examination revealed sheets of macrophages, plasma cells, lymphocytes, and occasional fibroblasts and neutrophils infiltrating the third eyelid stroma. A diagnosis of chronic granulomatous conjunctivitis was made. Grossly and histopathologically this case closely resembles previously described cases of nodular granulomatous conjunctivitis involving the third eyelids of Collie dogs. This report describes an unusual case of nodular granulomatous conjunctivitis isolated to the third eyelids in an Australian Kelpie. Resolution of the condition was achieved with a combination of oral doxycycline, niacinamide and prednisolone.
Surgical management of cicatricial ectropion following scarring dermatopathies in two dogs
Authors: Donaldson David , Smith Kerry M. , Shaw Stephen C. , Sansom Jane , Hartley Claudia
Published: September 2005

Two dogs were presented with severe dermatopathies leading to cicatricial ectropion and severe surface ocular pathology. In one case, idiopathic sterile pyogranuloma was diagnosed on histopathology and managed with systemic immunosuppressive therapy. In the second case, demodicosis was diagnosed on skin scrapings and successfully treated with acaricidal medication. Following control of the underlying dermatopathies, both cases developed cicatricial ectropion as a result of scarring and contracture of eyelid tissue. Wharton–Jones (V–Y) blepharoplasties were performed on the affected eyelids and resulted in improved eyelid function and reduced corneal exposure in both cases.
The α1D‐adrenoceptor antagonist BMY 7378 is also an α2C‐adrenoceptor antagonist
Authors: Cleary L. , Murad K. , Bexis S. , Docherty J. R.
Published: September 2005

1 We have investigated the actions of the α1D‐adrenoceptor selective antagonist BMY 7378 in comparison with yohimbine at α1‐ and α2‐adrenoceptors.

2 In rat aorta (α1D‐adrenoceptor), BMY 7378 (pA2 of 8.67) was about 100 times more potent than yohimbine (pA2 of 6.62) at antagonizing the contractile response to noradrenaline.

3 In human saphenous vein (α2C‐adrenoceptor), BMY 7378 (pA2 of 6.48) was approximately 10 times less potent than yohimbine (pA2 of 7.56) at antagonizing the contractile response to noradrenaline.

4 In prostatic portions of rat vas deferens, BMY 7378 (10 μm) did not significantly affect the concentration‐dependent inhibition of single pulse nerve stimulation‐evoked contractions by xylazine (an action at prejunctional α2D‐adrenoceptors).

5 In ligand‐binding studies, BMY 7378 showed 10‐fold selectivity for α2C‐adrenoceptors (pKi of 6.54) over other α2‐adrenoceptors.

6 It is concluded that BMY 7378, in addition to α1D‐adrenoceptor selectivity in terms of α1‐adrenoceptors, shows selectivity for α2C‐adrenoceptors in terms of α2‐adrenoceptors.
A novel mechanism prevents the development of hypertension during chronic cold stress
Authors: Westfall T. C. , Yang C.‐L. , Chen X. , Naes L. , Vickery L. , Macarthur H. , Han S.
Published: September 2005

1 Chronic cold exposure of rats (7 days in a cold room at 4 °C) attenuated the sympathetic nerve stimulation (NS)‐induced overflow of noradrenaline (NE) (measured by high‐performance liquid chromatography, coupled to electrochemical detection) appearing in the perfusate/superfusate of the perfused mesenteric arterial bed as well as the increase in the perfusion pressure.

2 The same type of cold exposure resulted in an increase in tyrosine hydroxylase (TH) gene expression measured in the superior cervical ganglion and NE content measured in the mesenteric artery obtained from cold‐exposed rats.

3 Addition of sodium nitroprusside, a nitric oxide (NO) donor, to the buffer perfusing the mesenteric arterial bed obtained from rats maintained at room temperature also resulted in an attenuation of the NS‐induced overflow of NE and increase in perfusion pressure.

4 
N
c‐nitro‐l‐arginine methyl ester (l‐NAME), an NO synthase inhibitor, placed in the drinking water prevented the attenuation of the pre‐ and post‐junctional responses to NS of the mesenteric arterial bed obtained from cold‐exposed rats.

5 
l‐NAME treatment also increased the cold‐induced elevation of blood pressure seen in whole animals.

6 The present results are consistent with the idea that cold exposure leads to a concomitant increase in sympathetic nerve activity and production of NO. We hypothesize that the increase in production and release of NO results in a decrease in the biologically active form of NE despite increased synthesis and release of the catecholamine.

7 It is concluded that the above‐mentioned interactions serve as a protective mechanism offsetting the increased release and action of NE from sympathetic nerves and thus preventing the development of hypertension.
Deception and Fear in Politically Oppressive Contexts: Its Trickle‐Down Effect on Families
Authors: Sluzki Carlos E.
Published: September 2005

The mystified reality, restricted options, and inherent risks of living in countries under a repressive political regime trigger survival tactics that reduce individual's reliance on social support, as well as semantic and cognitive restrictions and alternative codes, and silences that translate into symptoms. While this is the case for the average citizen, it is even more pronounced in individuals and families directly touched by the repressive apparatus. These processes are discussed and two clinical examples are provided to illustrate them.
Absence of Evidence for the Participation of the Macrophage Cellular Prion Protein in Infection with
Brucella suis
Authors: Fontes Pascaline , Alvarez-Martinez Maria-Teresa , Gross Antoine , Carnaud Claude , Köhler Stephan , Liautard Jean-Pierre
Published: September 2005

Brucella
spp. are stealthy bacteria that enter host cells without major perturbation. The molecular mechanism involved is still poorly understood, although numerous studies have been published on this subject. Recently, it was reported that
Brucella abortus
utilizes cellular prion protein (PrP
C
) to enter the cells and to reach its replicative niche. The molecular mechanisms involved were not clearly defined, prompting us to analyze this process using blocking antibodies against PrP
C
. However, the behavior of
Brucella
during cellular infection under these conditions was not modified. In a next step, the behavior of
Brucella
in macrophages lacking the prion gene and the infection of mice knocked out for the prion gene were studied. We observed no difference from results obtained with the wild-type control. Although some contacts between PrP
C
and
Brucella
were observed on the surface of the cells by using confocal microscopy, we could not show that
Brucella
specifically bound recombinant PrP
C
. Therefore, we concluded from our results that prion protein (PrP
C
) was not involved in
Brucella
infection.
Characterization of
Legionella pneumophila pmiA
, a Gene Essential for Infectivity of Protozoa and Macrophages
Authors: Miyake Masaki , Watanabe Takurou , Koike Hitomi , Molmeret Maëlle , Imai Yasuyuki , Abu Kwaik Yousef
Published: September 2005

The ability of
Legionella pneumophila
to cause pneumonia is dependent on intracellular replication within alveolar macrophages. The Icm/Dot secretion apparatus is essential for the ability of
L. pneumophila
to evade endocytic fusion, to remodel the phagosome by the endoplasmic reticulum (ER), and to replicate intracellularly. Protozoan and macrophage infectivity (
pmi
) mutants of
L. pneumophila
, which include 11
dot/icm
mutants, exhibit defects in intracellular growth and replication within both protozoa and macrophages. In this study we characterized one of the
pmi
loci,
pmiA
. In contrast to the parental strain, the
pmiA
mutant is defective in cytopathogenicity for protozoa and macrophages. This is a novel mutant that exhibits a partial defect in survival within U937 human macrophage-like cells but exhibits a severe growth defect within
Acanthamoeba polyphaga
, which results in elimination from this host. The intracellular defects of this mutant are complemented by the wild-type
pmiA
gene on a plasmid. In contrast to phagosomes harboring the wild-type strain, which exclude endosomal-lysosomal markers, the
pmiA
mutant-containing phagosomes acquire the late endosomal-lysosomal markers LAMP-1 and LAMP-2. In contrast to the parental strain-containing phagosomes that are remodeled by the ER, there was a decrease in the number of ER-remodeled phagosomes harboring the
pmiA
mutant. Among several
Legionella
species examined, the
pmiA
gene is specific for
L. pneumophila
. The predicted amino acid sequence of the PmiA protein suggests that it is a transmembrane protein with three membrane-spanning regions. PmiA is similar to several hypothetical proteins produced by bacteria with a type IV secretion apparatus. Importantly, the defect in
pmiA
abolishes the pore-forming activity, which has been attributed to the Icm/Dot type IV secretion system. However, the mutant is sensitive to NaCl, and this sensitivity is abrogated in the
icm/dot
mutants. These results suggest that PmiA is a novel virulence factor that is involved in intracellular survival and replication of
L. pneumophila
in macrophages and protozoan cells.
Requirement for Intercellular Adhesion Molecule 1 and Caveolae in Invasion of Human Oral Epithelial Cells by
Porphyromonas gingivalis
Authors: Tamai Riyoko , Asai Yasuyuki , Ogawa Tomohiko
Published: September 2005

Porphyromonas gingivalis
, a periodontopathic bacterium, is known to invade oral epithelial cells in periodontal lesions, although the mechanism is unclear. In the present study, goat polyclonal anti-intercellular adhesion molecule 1 (anti-ICAM-1) antibody inhibited the invasion of
P. gingivalis
into KB cells (human oral epithelial cells). Further, the
P. gingivalis
fimbria, a pathogenic adhesion molecule, bound to recombinant human ICAM-1, as shown by enzyme-linked immunosorbent assay.
P. gingivalis
was also found to colocalize with ICAM-1 on KB cells, as seen with an immunofluorescence microscope, and the knockdown of ICAM-1 in KB cells resulted in the inhibition of
P. gingivalis
invasion by RNA interference. In addition, methyl-β-cyclodextrin, a cholesterol-binding agent, inhibited the colocalization of
P. gingivalis
with ICAM-1 and invasion by the microorganism. The colocalization of caveolin-1, a caveolar marker protein, on KB cells with
P. gingivalis
was also shown, and the knockdown of caveolin-1 in KB cells caused a reduced level of
P. gingivalis
invasion. These results suggest that ICAM-1 and caveolae are required for the invasion of
P. gingivalis
into human oral epithelial cells, and these molecules appear to be associated with the primary stages of the development and progression of chronic periodontitis.
Infection withMycobacterium ulceransInduces Persistent Inflammatory Responses in Mice
Authors: Oliveira Martinha S. , Fraga Alexandra G. , Torrado Egídio , Castro António G. , Pereira João P. , Filho Adhemar Longatto , Milanezi Fernanda , Schmitt Fernando C. , Meyers Wayne M. , Portaels Françoise , Silva Manuel T. , Pedrosa Jorge
Published: September 2005

Buruli ulcer (BU) is a devastating, necrotizing, tropical skin disease caused by infections withMycobacterium ulcerans. In contrast to other mycobacterioses, BU has been associated with minimal or absent inflammation. However, here we show that in the mouseM. ulceransinduces persistent inflammatory responses with virulence-dependent patterns. Mycolactone-positive, cytotoxic strains are virulent for mice and multiply progressively, inducing both early and persistent acute inflammatory responses. The cytotoxicity of these strains leads to progressive destruction of the inflammatory infiltrates by postapoptotic secondary necrosis, generating necrotic acellular areas with extracellular bacilli released by the lysis of infected phagocytes. The necrotic areas, always surrounded by acute inflammatory infiltrates, expand through the progressive invasion of healthy tissues around the initial necrotic lesions by bacteria and by newly recruited acute inflammatory cells. Our observations show that the lack of inflammatory infiltrates in the extensive areas of necrosis seen in advanced infections results from the destruction of continuously produced inflammatory infiltrates and not fromM. ulcerans-induced local or systemic immunosuppression. Whether this is the mechanism behind the predominance of minimal or absent inflammatory responses in BU biopsies remains to be elucidated.
Alteration in Host Cell Tropism Limits the Efficacy of Immunization with a Surface Protein of Malaria Merozoites
Authors: Shi Qifang , Cernetich Amy , Daly Thomas M. , Galvan Gina , Vaidya Akhil B. , Bergman Lawrence W. , Burns James M.
Published: September 2005

Immunization withPlasmodium yoeliimerozoite surface protein-8 (PyMSP-8) has been shown to protect mice against lethalP. yoelii17XL malaria. Here we demonstrate that PyMSP-8-specific antibodies preferentially suppressP. yoelii17XL growth in mature erythrocytes compared to growth in reticulocytes and do not suppress the growth of nonlethalP. yoelii17X, a parasite that primarily replicates in reticulocytes. The protection against normocyte-associatedP. yoeliimalaria parasites is mediated by antibodies that recognize conformational epitopes of PyMSP-8 that are nonpolymorphic. We examined changes in gene expression in reticulocyte-restrictedP. yoelii17XL parasites that escaped neutralization by PyMSP-8-specific antibodies usingP. yoeliiDNA microarrays. Of interest,Pymsp-8gene expression decreased, while the expression ofmsp-1,msp-7, and several rhoptry protein genes increased. Breakthrough parasites also exhibited increases in the expression of a subset ofyirandPyst-agenes that are predicted to encode polymorphic antigens expressed on the surface of infected erythrocytes. These data suggest that changes in the expression of parasite proteins expressed on the merozoite surface, as well as the surface of infected erythrocytes, may alter host cell tropism and contribute to the ability of malaria parasites to evade merozoite-specific, neutralizing antibodies.
Doubly Branched Hexasaccharide Epitope on the Cell Wall Polysaccharide of Group A Streptococci Recognized by Human and Rabbit Antisera
Authors: Michon Francis , Moore Samuel L. , Kim John , Blake Milan S. , Auzanneau France-Isabelle , Johnston Blair D. , Johnson Margaret A. , Pinto B. Mario
Published: September 2005

A number of epitope specificities associated with the cell wall polysaccharide antigen of group A streptococci were identified in a polyclonal rabbit antiserum induced in rabbits by whole group A streptococci and in polyclonal convalescent human antisera from children that had recovered from streptococcal A infections. The identification was achieved by using a series of synthetic oligosaccharides, glycoconjugates, and bacterial polysaccharide inhibitors to inhibit the binding of the group A helical polysaccharide to the polyclonal antisera. The exclusively dominant epitope expressed in the convalescent human antisera was the doubly branched extended helical hexasaccharide with the structure α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap(1→3)α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap. The hexasaccharide epitope also bound with the highest immunoreactivity to the rabbit antiserum. In contrast, the human antisera did not show significant binding to the singly branched pentasaccharide with the structure α-l-Rhap(1→2)α-l-Rhap(1→3)α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhapor the branched trisaccharide α-l-Rhap(1→2)[β-d-GlcpNAc(1→3)]α-l-Rhap, although both these haptens bound significantly to the same rabbit antiserum, albeit with less immunoreactivity than the hexasaccharide. Inhibition studies using streptococcal group A and B rabbit antisera and the inhibitors indicated above also suggested that the group A carbohydrate, unlike the group B streptococcal polysaccharide, does not contain the disaccharide α-l-Rhap(1→2)α-l-Rhapmotif at its nonreducing chain terminus, stressing the importance of mapping the determinant specificities of these two important streptococcal subcapsular group polysaccharides to fully understand the serological relationships between group A and group B streptococci.
Minimum Infective Dose of
Mycobacterium bovis
in Cattle
Authors: Dean Gillian S. , Rhodes Shelley G. , Coad Michael , Whelan Adam O. , Cockle Paul J. , Clifford Derek J. , Hewinson R. Glyn , Vordermeier H. Martin
Published: September 2005

The aim of this work was to determine the minimum infective dose of
Mycobacterium bovis
necessary to stimulate specific immune responses and generate pathology in cattle. Four groups of calves (20 animals) were infected by the intratracheal route with 1,000, 100, 10, or 1 CFU of
M. bovis
. Specific immune responses (gamma interferon [IFN-γ] and interleukin-4 [IL-4] responses) to mycobacterial antigens were monitored throughout the study, and the responses to the tuberculin skin test were assessed at two times. Rigorous post mortem examinations were performed to determine the presence of pathology, and samples were taken for microbiological and histopathological confirmation of
M. bovis
infection. One-half of the animals infected with 1 CFU of
M. bovis
developed pulmonary pathology typical of bovine tuberculosis. No differences in the severity of pathology were observed for the different
M. bovis
doses. All animals that developed pathology were skin test positive and produced specific IFN-γ and IL-4 responses. No differences in the sizes of the skin test reactions, the times taken to achieve a positive IFN-γ result, or the levels of the IFN-γ and IL-4 responses were observed for the different
M. bovis
doses, suggesting that diagnostic assays (tuberculin skin test and IFN-γ test) can detect cattle soon after
M. bovis
infection regardless of the dose. This information should be useful in modeling the dynamics of bovine tuberculosis in cattle and in assessing the risk of transmission.
Legionella pneumophila
NudA Is a Nudix Hydrolase and Virulence Factor
Authors: Edelstein Paul H. , Hu Baofeng , Shinzato Takashi , Edelstein Martha A. C. , Xu Wenlian , Bessman Maurice J.
Published: September 2005

We studied the identity and function of the 528-bp gene immediately upstream of
Legionella pneumophila
F2310
ptsP
(enzyme I
Ntr
). This gene,
nudA
, encoded for a Nudix hydrolase based on the inferred protein sequence. NudA had hydrolytic activity typical of other Nudix hydrolases, such as
Escherichia coli
YgdP, in that Ap

n

A’s, in particular diadenosine pentaphosphate (Ap
5
A), were the preferred substrates. NudA hydrolyzed Ap
5
A to ATP plus ADP. Both
ptsP
and
nudA
were cotranscribed. Bacterial two-hybrid analysis showed no PtsP-NudA interactions. Gene
nudA
was present in 19 of 20 different
L. pneumophila
strains tested and in 5 of 10 different
Legionella
spp. other than
L. pneumophila
. An in-frame
nudA
mutation was made in
L. pneumophila
F2310 to determine the phenotype. The
nudA
mutant was an auxotroph that grew slowly in liquid and on solid media and had a smaller colony size than its parent. In addition, the mutant was more salt resistant than its parent and grew very poorly at 25°C; all of these characteristics, as well as auxotrophy and slow-growth rate, were reversed by transcomplementation with
nudA
. The
nudA
mutant was outcompeted by about fourfold by the parent in competition studies in macrophages; transcomplementation almost completely restored this defect. Competition studies in guinea pigs with
L. pneumophila
pneumonia showed that the
nudA
mutant was outcompeted by its parent in both lung and spleen. NudA is of major importance for resisting stress in
L. pneumophila
and is a virulence factor.
Antigen Requirements for Efficient Priming of CD8+T Cells byLeishmania major-Infected Dendritic Cells
Authors: Bertholet Sylvie , Debrabant Alain , Afrin Farhat , Caler Elisabeth , Mendez Susana , Tabbara Khaled S. , Belkaid Yasmine , Sacks David L.
Published: September 2005

CD4+and CD8+T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasiteLeishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential ofL. majortransgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4+or OT-I CD8+T cells. Truncated OVA was expressed inL. majoras part of a secreted or nonsecreted chimeric protein withL. donovani3′ nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected withL. majorthat secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments inL. majorand suggest that proteins secreted or released byL. majorin infected DC are a major source of peptides for the generation of parasite-specific CD8+T cells. The ability ofL. majortransgenic parasites to activate OT-I CD8+T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level.
Analysis of the Behavior of
eryC
Mutants of
Brucella suis
Attenuated in Macrophages
Authors: Burkhardt Sonja , Jiménez de Bagüés Maria P. , Liautard Jean-Pierre , Köhler Stephan
Published: September 2005

The facultatively intracellular pathogen
Brucella
, characterized by its capacity to replicate in professional and non professional phagocytes, also causes abortion in ruminants. This property has been linked to the presence of erythritol in the placenta, as brucellae preferentially utilize erythritol. The
ery
operon encodes enzymes involved in erythritol metabolism, and a link with virulence has since been discussed. Allelic exchange mutants in
eryC
of
Brucella suis
were erythritol sensitive in vitro with a MIC of 1 to 5 mM of erythritol. Their multiplication in macrophage-like cells was 50- to 90-fold reduced, but complementation of the mutant restored wild-type levels of intracellular multiplication and the capacity to use erythritol as a sole carbon source. In vivo, the
eryC
mutant colonized the spleens of infected BALB/c mice to a significantly lower extent than the wild type and the complemented strain. Interestingly,
eryC
mutants that were in addition spontaneously erythritol tolerant nevertheless exhibited wild-type-like intramacrophagic and intramurine replication. We concluded from our results that erythritol was not an essential carbon source for the pathogen in the macrophage host cell but that the inactivation of the
eryC
gene significantly reduced the intramacrophagic and intramurine fitness of
B. suis
.
Antibodies to the Iron Uptake ABC Transporter Lipoproteins PiaA and PiuA Promote Opsonophagocytosis of
Streptococcus pneumoniae
Authors: Jomaa Maha , Yuste Jose , Paton James C. , Jones Christopher , Dougan Gordon , Brown Jeremy S.
Published: September 2005

PiaA and PiuA are the lipoprotein components of the Pia and Piu
Streptococcus pneumoniae
iron uptake ABC transporters and are required for full virulence in mouse models of infection. Active or passive vaccination with recombinant PiuA and PiaA protects mice against invasive
S. pneumoniae
disease. In this study we have analyzed the antibody responses and mechanism of protection induced by PiuA and PiaA in more detail. For both proteins, two booster vaccinations induced stronger antibody responses in mice than a single or no booster vaccinations, and 5 μg of protein induced similar levels of antibody responses as 20 μg. Immunoglobulin G (IgG) subclass-specific enzyme-linked immunosorbent assays demonstrated that the antibody response to PiuA and PiaA was predominantly IgG1, with induction of only low levels of IgG2a. Anti-PiaA and anti-PiuA polyclonal rabbit antibodies bound to the surface of live
S. pneumoniae
when assessed by flow cytometry but did not inhibit growth of
S. pneumoniae
in cation-depleted medium or bacterial susceptibility to the iron-dependent antibiotic streptonigrin. However, anti-PiaA and anti-PiuA did increase complement-independent and -dependent opsonophagocytosis of different serotypes of
S. pneumoniae
by the human neutrophil cell line HL60. Hence, vaccination with PiaA and PiuA protects against
S. pneumoniae
infection by inducing antibodies that promote bacterial opsonophagocytosis rather than inhibiting iron transport.
Homologues of Insecticidal Toxin Complex Genes in
Yersinia enterocolitica
Biotype 1A and Their Contribution to Virulence
Authors: Tennant Sharon M. , Skinner Narelle A. , Joe Angela , Robins-Browne Roy M.
Published: September 2005

Yersinia enterocolitica
is an enteric pathogen that consists of six biotypes: 1A, 1B, 2, 3, 4, and 5. Strains of the latter five biotypes can carry a virulence plasmid, known as pYV, and several well-characterized chromosomally encoded virulence determinants.
Y. enterocolitica
strains of biotype 1A lack the virulence-associated markers of pYV-bearing strains and were once considered to be avirulent. There is growing epidemiological, clinical, and experimental evidence, however, to suggest that some biotype 1A strains are virulent and can cause gastrointestinal disease. To identify potential virulence genes of pathogenic strains of
Y. enterocolitica
biotype 1A, we used genomic subtractive hybridization to determine genetic differences between two biotype 1A strains: an environmental isolate,
Y. enterocolitica
IP2222, and a clinical isolate,
Y. enterocolitica
T83. Among the
Y. enterocolitica
T83-specific genes we identified were three,
tcbA
,
tcaC
, and
tccC
, that showed homology to the insecticidal toxin complex (TC) genes first discovered in
Photorhabdus luminescens
. The
Y. enterocolitica
T83 TC gene homologues were expressed by
Y. enterocolitica
T83 and were significantly more prevalent among clinical biotype 1A strains than other
Yersinia
isolates. Inactivation of the TC genes in
Y. enterocolitica
T83 resulted in mutants which were attenuated in the ability to colonize the gastrointestinal tracts of perorally infected mice. These results indicate that products of the TC gene complex contribute to the virulence of some strains of
Y. enterocolitica
biotype 1A, possibly by facilitating their persistence in vivo.
Biochemical Characterization and Vaccine Potential of a Heme-Binding Glutathione Transferase from the Adult Hookworm
Ancylostoma caninum
Authors: Zhan Bin , Liu Sen , Perally Samirah , Xue Jian , Fujiwara Ricardo , Brophy Peter , Xiao Shuhua , Liu Yueyuan , Feng Jianjun , Williamson Angela , Wang Yan , Bueno Lilian L. , Mendez Susana , Goud Gaddam , Bethony Jeffrey M. , Hawdon John M. , Loukas Alex , Jones Karen , Hotez Peter J.
Published: September 2005

We report the cloning and expression of
Ac
-GST-1, a novel glutathione
S
-transferase from the adult hookworm
Ancylostoma caninum
, and its possible role in parasite blood feeding and as a vaccine target. The predicted
Ac
-GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding
Ac
-GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein,
Ac
-GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant
Ac
-GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of
Ac
-GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with
Ac
-GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with
Ac
-GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous
Necator americanus
larvae. These studies suggest that
Ac
-GST-1 is a possible drug and vaccine target for hookworm infection.
Innate Refractoriness of the Lewis Rat to Toxoplasmosis Is a Dominant Trait That Is Intrinsic to Bone Marrow-Derived Cells
Authors: Sergent Véronique , Cautain Bastien , Khalife Jamal , Deslée Didier , Bastien Patrick , Dao Anne , Dubremetz Jean-François , Fournié Gilbert J. , Saoudi Abdelhadi , Cesbron-Delauw Marie-France
Published: September 2005

Toxoplasmosis is a ubiquitous parasitic infection causing a wide spectrum of diseases. It is usually asymptomatic but can lead to severe ocular and neurological disorders. Among the small-animal models available to study factors that determine susceptibility to toxoplasmosis, the rat appears to be rather similar to humans, particularly in terms of resistance to acute infection. Here, we demonstrate that the Lewis (LEW) rat strain displays an unexpected refractoriness toToxoplasmainfection. Complete resistance was assessed by both negative anti-Toxoplasmaserology and lack of detection of the parasite during the course of infection. In this model, sex, age, major histocompatibility complex, and inoculum size had no effect on resistance. Interestingly, progeny from F1hybrid crosses between Fischer (F344) or Brown Norway susceptible rats and LEW resistant rats were also fully resistant, showing a dominant effect of the gene or set of genes. Furthermore, resistance of the LEW rat was shown to be dependent on hematopoietic cells and partially abrogated by neutralization of endogenous gamma interferon. To our knowledge, this is the first observation of a rodent strain that is refractory toToxoplasmainfection. This model is therefore an attractive and powerful tool to dissect host genetic factors involved in susceptibility to toxoplasmosis.
Pathophysiological Manifestations in Mice Exposed to Anthrax Lethal Toxin
Authors: Culley Nathan C. , Pinson David M. , Chakrabarty Anuradha , Mayo Matthew S. , LeVine Steven M.
Published: September 2005

Pathophysiological changes associated with anthrax lethal toxin included loss of plasma proteins, decreased platelet count, slower clotting times, fibrin deposits in tissue sections, and gross and histopathological evidence of hemorrhage. These findings suggest that blood vessel leakage and hemorrhage lead to disseminating intravascular coagulation and/or circulatory shock as an underlying pathophysiological mechanism.
Protective Role of Neutrophils in Mice Experimentally Infected with
Rhodococcus equi
Authors: Martens Ronald J. , Cohen Noah D. , Jones Samuel L. , Moore Thomas A. , Edwards John F.
Published: September 2005

Neutrophils are important in controlling early infections with the intracellular bacterium
Rhodococcus equi
. Antineutrophil monoclonal antibody (RB6-8C5)-induced neutrophil deficiency during the first week after experimental infection of mice with
R. equi
resulted in more severe disease and significantly increased tissue concentrations of
R. equi.
Relationship of Virulence Factor Expression to Evolved Virulence in Mouse-Passaged
Cryptococcus neoformans
Lines
Authors: McClelland Erin E. , Perrine Wesley T. , Potts Wayne K. , Casadevall Arturo
Published: September 2005

Serial passage of
Cryptococcus neoformans
in mice increases virulence relative to the nonpassaged line. Postpassaged lines showed no difference in the expression of most known virulence factors, with the exception that the more virulent lines had smaller capsules in vitro. These data imply that other mechanisms of virulence remain to be discovered.
Effects of Dexamethasone and Transient Malnutrition on Rabbits Infected with AerosolizedMycobacterium tuberculosisCDC1551
Authors: Kesavan Anup K. , Mendez Susana E. , Hatem Christine L. , Lopez-Molina Javier , Aird Katherine , Pitt M. Louise M. , Dannenberg Arthur M. , Manabe Yukari C.
Published: September 2005

Malnutrition is common in the developing world, where tuberculosis is often endemic. Rabbits infected with aerosolizedMycobacterium tuberculosisthat subsequently became inadvertently and transiently malnourished had compromised cell-mediated immunity comparable to that of the rabbits immunosuppressed with dexamethasone. They had significant leukopenia and reduced delayed-type hypersensitivity responses. Malnutrition dampened cell-mediated immunity and would interfere with diagnostic tests that rely on intact CD4 T-cell responses.
Metabolic and endothelial effects of trimetazidine on forearm skeletal muscle in patients with type 2 diabetes and ischemic cardiomyopathy
Authors: Monti Lucilla D. , Setola Emanuela , Fragasso Gabriele , Camisasca Riccardo P. , Lucotti Pietro , Galluccio Elena , Origgi Anna , Margonato Alberto , Piatti PierMarco
Published: September 2005

The aim of the present study was to evaluate the effect of prolonged inhibition of β-oxidation on glucose and lipid muscle forearm metabolism and cGMP and endothelin-1 forearm release in patients with type 2 diabetes mellitus and ischemic cardiomyopathy. Fifteen patients were randomly allocated in a double-blind cross-over parallel study with trimetazidine (20 mg tid) or placebo lasting 15 days. At the end of each period, all patients underwent euglycemic hyperinsulinemic clamps with forearm indirect calorimetry and endothelial balance of vasodilator and vasoconstricor factors. Compared with placebo, trimetazidine induced 1) an increase in insulin-induced forearm glucose uptake and glucose oxidation accompained by a reduction in forearm lipid oxidation and citrate release and 2) a decrease of endothelin-1 release paralleled by a significant increase in forearm cGMP release. Forearm glucose oxidation significantly correlated with cGMP release ( r = 0.37, P < 0.04), whereas forearm lipid oxidation positively correlated with endothelin-1 release ( r = 0.40, P < 0.03). In conclusion, for the first time, we demonstrated that insulin-induced forearm glucose oxidation and forearm cGMP release were increased whereas forearm endothelin-1 release was decreased during trimetazidine treatment. Muscle's metabolic and vascular effects of trimetazidine add new interest in the use of trimetazidine in type 2 diabetic patients with cardiovascular disease.
Growth hormone receptor deficiency results in blunted ghrelin feeding response, obesity, and hypolipidemia in mice
Authors: Egecioglu Emil , Bjursell Mikael , Ljungberg Anna , Dickson Suzanne L. , Kopchick John J. , Bergström Göran , Svensson Lennart , Oscarsson Jan , Törnell Jan , Bohlooly-Y Mohammad
Published: September 2005

We have previously shown that growth hormone (GH) overexpression in the brain increased food intake, accompanied with increased hypothalamic agouti-related protein (AgRP) expression. Ghrelin, which stimulates both appetite and GH secretion, was injected intracerebroventricularly to GHR−/−and littermate control (+/+) mice to determine whether ghrelin's acute effects on appetite are dependent on GHR signaling. GHR−/−mice were also analyzed with respect to serum levels of lipoproteins, apolipoprotein (apo)B, leptin, glucose, and insulin as well as body composition. Central injection of ghrelin into the third dorsal ventricle increased food consumption in +/+ mice, whereas no change was observed in GHR−/−mice. After ghrelin injection, AgRP mRNA expression in the hypothalamus was higher in +/+ littermates than in GHR−/−mice, indicating a possible importance of AgRP in the GHR-mediated effect of ghrelin. Compared with controls, GHR−/−mice had increased food intake, leptin levels, and total and intra-abdominal fat mass per body weight and deceased lean mass. Moreover, serum levels of triglycerides, LDL and HDL cholesterol, and apoB, as well as glucose and insulin levels were lower in the GHR−/−mice. In summary, ghrelin's acute central action to increase food intake requires functionally intact GHR signaling. Long-term GHR deficiency in mice is associated with high plasma leptin levels, obesity, and increased food intake but a marked decrease in all lipoprotein fractions.
gp100209–2M Peptide Immunization of Human Lymphocyte Antigen-A2+ Stage I-III Melanoma Patients Induces Significant Increase in Antigen-Specific Effector and Long-Term Memory CD8+ T Cells
Authors: Walker Edwin B. , Haley Daniel , Miller William , Floyd Kevin , Wisner Ketura P. , Sanjuan Nelson , Maecker Holden , Romero Pedro , Hu Hong-Ming , Alvord W. Gregory , Smith John W. , Fox Bernard A. , Urba Walter J.
Published: September 2005

Thirty-five HLA-A2+ patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100209–2M, emulsified in Montanide adjuvant. Direct ex vivo gp100209–2M tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer+ CD8+ T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05–8.9%). Ex vivo IFN-γ cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100209–2M in vitro activation showed that for all of the patients studied tetramer+ CD8+ T cells produced IFN-γ; however, some patients had significant numbers of tetramer+ IFN-γ− CD8+T cells suggesting functional anergy. Additionally, 8 day gp100209–2M in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer+ CD8+ T cells from postvaccine cells for 34 patients, and these IVS tetramer+ CD8+ T cells were functionally responsive by IFN-γ cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8+ T cells demonstrated the proliferation of functionally anergic gp100209–2M- tetramer+ CD8+ T cells in several patients and also indicated interpatient variability of gp100209–2M stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer+ CD8+ T cells (78.1 ± 4.2%) had either an “effector” (CD45 RA+/CCR7−) or an “effector-memory” phenotype (CD45RA−/CCR7−). Notably, analysis of PBMCs collected 12–24 months after vaccine therapy demonstrated the durable presence of gp100209–2M-specific memory CD8+ T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8+ T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.
Elevated Cyclooxygenase-2 Expression Is Associated with Altered Expression of p53 and SMAD4, Amplification of HER-2/neu, and Poor Outcome in Serous Ovarian Carcinoma
Authors: Erkinheimo Tiina-Liisa , Lassus Heini , Finne Patrik , van Rees Bastiaan P. , Leminen Arto , Ylikorkala Olavi , Haglund Caj , Butzow Ralf , Ristimäki Ari
Published: September 2005

Purpose and Experimental Design: Cyclooxygenase-2 (COX-2) is frequently expressed in human adenocarcinomas and inhibition of COX-2 suppresses tumor formation in various animal models of carcinogenesis. We analyzed expression of COX-2 protein in human serous ovarian carcinomas by immunohistochemistry (n = 442) and by Western blotting (n = 12) and COX-2 mRNA by reverse transcriptase PCR (n = 12). COX-2 immunoreactivity was correlated to clinicopathological variables and to expression of p53 and SMAD4 as detected by immunohistochemistry and to amplification of HER-2/neu as detected by in situ hybridization.

Results: COX-2 mRNA expression was detected in 75% (9 of 12) and COX-2 protein in 42% (5 of 12) of the serous ovarian adenocarcinoma specimens as detected by reverse transcriptase-PCR and Western blot analysis, respectively. Moderate to strong (elevated) immunoreactivity for COX-2 was detected in 70% (310 of 442) of the tumors. Elevated COX-2 expression associated with reduced disease-specific survival (P = 0.0011), high histological grade (P < 0.0001), residual tumor size > 1 cm (P = 0.0111), and age > 57 years (P = 0.0099). Tumors with altered immunostaining pattern for p53 or SMAD4 expressed more frequently elevated levels of COX-2 when compared with the tumors with normal staining pattern of these tumor suppressor genes (P < 0.0001 and P = 0.0004, respectively). In addition, elevated COX-2 expression associated with amplification of HER-2/neu oncogene (P = 0.0479).

Conclusions: Our results suggest that elevated expression of COX-2 associates with reduced survival in serous ovarian carcinomas and that expression of COX-2 may be induced in these tumors by loss of tumor suppressor genes such as p53and SMAD4and by amplification of HER-2/neuoncogene.
Identification of Necrosis-Associated Genes in Glioblastoma by cDNA Microarray Analysis
Authors: Raza Shaan M. , Fuller Gregory N. , Rhee Chang Hun , Huang Suyun , Hess Kenneth , Zhang Wei , Sawaya Raymond
Published: September 2005

Purpose: In the field of cancer research, there has been a paucity of interest in necrosis, whereas studies focusing on apoptosis abound. In neuro-oncology, this is particularly surprising because of the importance of necrosis as a hallmark of glioblastoma (GBM), the most malignant and most common primary brain tumor, and the fact that the degree of necrosis has been shown to be inversely related to patient survival. It is therefore of considerable interest and importance to identify genes and gene products related to necrosis formation.

Experimental Design: We used a nylon cDNA microarray to analyze mRNA expression of 588 universal cellular genes in 15 surgically resected human GBM samples with varying degrees of necrosis. Gene expression was correlated with the degree of necrosis using rank correlation coefficients. The expression of identified genes was compared with their expression in tissue samples from 5 anaplastic astrocytomas (AAs). Immunostaining was used to determine whether genes showing the most positive correlation with necrosis were increasingly expressed in tumor tissues, as grade of necrosis increased.

Results: The hybridization results indicated that 26 genes showed significant correlation with the amount of necrosis. All 26 genes had functions associated with either Ras, Akt, tumor necrosis factor α, nuclear factor κB, apoptosis, procoagulation, or hypoxia. Nine genes were positively correlated with necrosis grade, and 17 genes were negatively correlated with necrosis grade. There were significant differences in the median expression levels of 3 of the 26 genes between grade III necrosis GBM and anaplastic astrocytoma (AA) samples; all but 1 of the genes had elevated expression when comparing necrosis grade III with AA samples. Two factors, the ephrin type A receptor 1 and the prostaglandin E2 receptor EP4 subtype, not previously considered in this context, were highlighted because of their particularly high (positive) correlation coefficients; immunostaining showed the products of these two genes to be localized in perinecrotic and necrotic regions and to be overexpressed in grade III GBMs, but not AAs. These two molecules also showed significant correlation with survival of GBM patients (P = 0.0034) in a combined model.

Conclusions: The application of cDNA expression microarray analysis has identified specific genes and patterns of gene expression that may help elucidate the molecular basis of necrogenesis in GBM. Additional studies will be required to further investigate and confirm these findings.