bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation of testicular artery Doppler velocimetry with kinetics and morphologic characteristics of epididymal sperm in dogs
Authors: Trautwein Luiz Guilherme Corsi , Souza Anne Kemmer , Cardoso Guilherme Schiess , da Costa Flaiban Karina Keller Marques , de Oliveira Dearo Antonio Cezar , Martins Maria Isabel Mello
Published: March 2020

Sperm quality can be affected by a reduction in testicular blood flow, which can be measured by Doppler ultrasonography. The aim of this study was to correlate the Doppler velocimetry of the testicular artery with kinetics of the epididymal spermatozoa in dogs. Twenty‐two dogs (44 testicles) were evaluated by Doppler ultrasonography in five regions of the testicular artery before orchiectomy. Spermatozoa were recovered by the epididymal tail compression technique and analysed for kinetics on a computer‐assisted semen analysis (CASA system). Morphology (modified Karras) and sperm membrane integrity were analysed by eosin–nigrosine staining. Data were analysed by Pearson's correlation test (p < .01). The mean total motility was 69.0% ± 17.7, progressive motility was 43.7% ± 14.7, average path velocity (VAP) was 127.0 µm/s ± 20.7, curvilinear velocity (VCL) was 221.0 µm/s ± 31.1, and sperm velocity index (SVI) was 389.9 ± 56.1. There were positive correlations between the peak systolic velocity (PSV) in the proximal supratesticular region with the SVI (r = .529), VCL (r = .555) and VAP (r = .473), and a negative correlation with the percentage of slow spermatozoa (r = −.463). The results suggest that the testicular artery blood flow velocity can positively affect the speed of spermatozoa movement. For the first time, we have correlated sperm kinetics with the Doppler evaluation of the testicular artery in dogs.
Dealing with the wicked issue of child poverty: Inter‐organizational networks as forums for collective debate and reflection
Authors: Jacquet Nicolas , Van Haute Dorien , De Corte Joris , Nisen Laurent , Vandenbroeck Michel , Roets Griet
Published: March 2020

In the international realm, inter‐organizational networking is perceived as a highly relevant instrument in social policy that enables welfare organizations to deal with “wicked issues.” In this article, we discuss the central empirical findings acquired from a recent qualitative research project that focuses on inter‐organizational networks that were formed at the local level to deal with the wicked issue of child poverty as a complex and multidimensional social problem. We explore how the network discussions about normative value orientations in four inter‐organizational networks evolve, and identify three central fields of tension that illustrate the complexity for local welfare actors in and across networks to create network strategies in dealing with child poverty: (a) selective versus universal provision, (b) conditional versus unconditional strategies, and (c) instrumental versus lifeworld‐oriented approaches. Our findings show that networks can function as valuable forums for collective debate and reflection, since different approaches and perspectives to tackle the problem of child poverty can be confronted with each other. Creating such a forum has the potential to challenge dominant conceptualizations and undesirable assumptions of complex social problems that are present in welfare practices and policies.
Nondestructive characterization of nanoscale subsurface features fabricated by selective etching of multilayered nanowire test structures using Mueller matrix spectroscopic ellipsometry based scatterometry
Authors: Korde Madhulika , Kal Subhadeep , Alix Cheryl , Keller Nick , Antonelli G. Andrew , Mosden Aelan , Diebold Alain C.
Published: March 2020

Nondestructive measurement of three-dimensional subsurface features remains one of the most difficult and unmet challenges faced during the fabrication of three-dimensional transistor architectures, especially nanosheet and nanowire based field effect transistors. The most critical fabrication step is the selective etching of silicon-germanium subsurface layers. The resulting shape and dimensions of the remaining Si(1 − x)Gex structure strongly impacts further processing steps and ultimately the electrical performance of gate-all-around transistors, thus creating the need for accurate inline metrology. In order to demonstrate the ability to characterize this etch, nanowire test structures made from Si(1 − x)Gex/Si/Si(1 − x)Gex/Si/Si(1 − x)Gex/Si multilayers have been characterized using Mueller matrix spectroscopic ellipsometry based scatterometry. Transmission electron microscopy images were used to corroborate the authors’ scatterometry measurements. Here, they successfully demonstrate the ability to measure the Si(1 − x)Gex etch, providing an industrially viable technique for inline three-dimensional metrology.
An Examination of the Relationship Between Motivation, Physical Activity, and Wearable Activity Monitor Use
Authors: Friel Ciarán P. , Garber Carol Ewing
Published: March 2020

Wearable physical activity (PA) monitors are widely promoted as a means to motivate people to be more active, but the motivational profile of users has never been assessed. This study’s purpose was to classify adult users by their motivational regulation scores and examine how these profiles were associated with moderate to vigorous PA (MVPA). Current users (N = 320) recruited across the United States completed a Web-based survey. Motivational regulations were measured using the Behavioral Regulation in Exercise Questionnaire, and associations with MVPA were explored using bivariate correlations. MVPA was more highly correlated with autonomous rather than controlling regulations. A cluster analysis was conducted using the respondents’ motivational regulation scores. Five motivational profiles emerged from this analysis, and they differed significantly across motivation and MVPA scores. PA monitor users characterized by more autonomous motives presented with higher MVPA. As technology use increases, assessing the multidimensionality of PA monitor users’ motivation may add value when researching PA behaviors.
The Iron Deficiency Response of Corynebacterium glutamicum and a Link to Thiamine Biosynthesis
Authors: Küberl Andreas , Mengus-Kaya Aliye , Polen Tino , Bott Michael
Published: March 2020

Iron is an essential element for most organisms but causes problems due to poor solubility under oxic conditions and due to toxicity by catalyzing the formation of reactive oxygen species (ROS). Therefore, bacteria have evolved complex regulatory networks for iron homeostasis aiming at a sufficient iron supply while minimizing ROS formation. In our study, the responses of the actinobacterium
Corynebacterium glutamicum
to iron limitation were analyzed, resulting in a detailed view on the processes involved in iron homeostasis in this model organism. In particular, we provide evidence that iron limitation causes TPP deficiency, presumably due to insufficient activity of the iron-dependent phosphomethylpyrimidine synthase (ThiC). TPP deficiency was deduced from the upregulation of genes controlled by a TPP riboswitch and secretion of metabolites caused by insufficient activity of the TPP-dependent enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. To our knowledge, the link between iron starvation and thiamine synthesis has not been elaborated previously.
Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes
Authors: Li Kaihuai , Hou Rongxian , Xu Huiyong , Wu Guichun , Qian Guoliang , Wang Haihong , Liu Fengquan
Published: March 2020

Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in
Lysobacter enzymogenes
. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in
L. enzymogenes
. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in
L. enzymogenes
. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.
The Biphasic mRNA Expression Pattern of Bovine Interleukin-8 inPasteurella haemolyticaLipopolysaccharide-Stimulated Alveolar Macrophages Is Primarily due to Tumor Necrosis Factor Alpha
Authors: Lafleur Rhonda L. , Abrahamsen Mitchell S. , Maheswaran Samuel K.
Published: March 2020

Pasteurella haemolyticaserotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) fromP. haemolyticain the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression inP. haemolyticaLPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P< 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-α) in the second phase of IL-8 mRNA expression. Our findings support a role forP. haemolyticaLPS and TNF-α in the induction of IL-8 from bovine alveolar macrophages.
A Peptide Domain on Gingipain R Which Confers Immunity against
Porphyromonas gingivalis
Infection in Mice
Authors: Genco Caroline Attardo , Odusanya Basil Michael , Potempa Jan , Mikolajczyk-Pawlinska Jowita , Travis James
Published: March 2020

The cysteine proteinases referred to as gingipains R (gingipain R1 and gingipain R2) and gingipain K produced by
Porphyromonas gingivalis
are virulence factors of this periodontal pathogen which likely act by interrupting host defense mechanisms and by participating in the penetration and destruction of host connective tissue. To examine the effect of immunization with gingipains R on the ability of
P. gingivalis
to colonize and invade in the mouse chamber model, BALB/c mice were immunized intraperitoneally with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or multiple antigenic peptide (MAP)-conjugated gingipain R-derived peptides and then challenged with
P. gingivalis
. Immunization of mice with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or a peptide derived from the N-terminal sequence of the catalytic domain of gingipains R (peptide A) followed by challenge with
P. gingivalis
A7436 resulted in protection from
P. gingivalis
invasion. In contrast, immunization with peptides corresponding to either a sequence encompassing the catalytic cysteine residue of gingipains R (peptide B) or an identical sequence within the catalytic domains of gingipain R1 and gingipain K (peptide C), followed by challenge with
P. gingivalis
, did not protect animals, nor did immunization with a peptide corresponding to sequences within the adhesion/hemagglutinin domain of gingipain R1 (peptide D) which have been shown to be directly involved in the hemagglutinin activity of gingipain R1. However, the immunoglobulin G (IgG) titer obtained following immunization with peptide D was comparable to that obtained following immunization with the N-terminal peptide (peptide A). Competitive enzyme-linked immunosorbent assays, using either the 95-kDa gingipain R1 or gingipain K as the competing soluble antigen, indicated that 42 and 53% of the antibodies induced by immunization with heat-killed bacteria recognize gingipain R1 and gingipain K, respectively; however, even at very high concentrations, the 50-kDa gingipain R2 did not hinder IgG binding to
P. gingivalis
. These results indicate that antibodies directed to the amino-terminal region of the catalytic domain of gingipains R are capable of inducing a protective immune response against
P. gingivalis
infection in the mouse chamber model.
Hemolytically Active (Acylated) Alpha-Hemolysin Elicits Interleukin-1β (IL-1β) but Augments the Lethality ofEscherichia coliby an IL-1- and Tumor Necrosis Factor-Independent Mechanism
Authors: Gleason Thomas G. , Houlgrave C. Webster , May Addison K. , Crabtree Traves D. , Sawyer Robert G. , Denham Woody , Norman James G. , Pruett Timothy L.
Published: March 2020

Many pathogenicEscherichia coliproduce the toxin alpha-hemolysin (Hly), and lipopolysaccharide (LPS), interleukin-1 (IL-1), and tumor necrosis factor (TNF) have all been recognized as important effector molecules during infections by gram-negative organisms. Despite the characterization of many in vitro effects of hemolysin, no direct relationship has been established between hemolysin, LPS, proinflammatory cytokine production, andE. coli-induced mortality. Previously, we have shown in vivo that hemolysin elicits a distinct IL-1α spike by 4 h into a lethal hemolyticE. coliinfection. Using three transformedE. colistrains, WAF108, WAF270, and WAH540 (which produce no Hly [Hlynull], acylated Hly [Hlyactive], or nonacylated Hly [Hlyinactive], respectively), we sought to determine the specific roles of hemolysin acylation, LPS, IL-1, and TNF in mediating the lethality ofE. coliinfection in mice. WAF270 was 100% lethal in BALB/c, C3H/HeJ, and C57BL/6 mice; in mice pretreated with antibody to the type 1 IL-1 receptor; in type 1 IL-1 receptor-deficient mice; and in dual (type 1 IL-1 receptor-type 1 TNF receptor)-deficient mice at doses which were nonlethal (0%) with both WAF108 and WAH540. At lethal doses, WAF270 killed by 6 ± 2.3 h while WAF108 and WAH540 killed at 36 ± 9.4 and 36 ± 13.8 h, respectively. These differences in mortality were not due to IL-1 or TNF release, and the enhanced expression of LPS, which corresponded to Hly expression, was not likely the primary factor causing mortality. We demonstrate that bacterial fatty acid acylation of hemolysin is required in order for it to elicit IL-1 release by monocytes and to confer its virulence onE. coli.
Involvement of the
Haemophilus ducreyi gmhA
Gene Product in Lipooligosaccharide Expression and Virulence
Authors: Bauer Beth A. , Stevens Marla K. , Hansen Eric J.
Published: March 2020

The lipooligosaccharide (LOS) present in the outer membrane of
Haemophilus ducreyi
is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in
H. ducreyi
35000 was found to encode a predicted protein that had 87% identity with the protein product of the
gmhA
(
isn
) gene of
Haemophilus influenzae
. In
H. influenzae
type b, inactivation of the
gmhA
gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396–402, 1996). The
H. ducreyi gmhA
gene was able to complement a
gmhA
-deficient
Escherichia coli
strain, a result which confirmed the identity of this gene. When the
gmhA
gene of
H. ducreyi
was inactivated by insertion of a
cat
cartridge, the resultant
H. ducreyi gmhA
mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type
H. ducreyi
strain and its isogenic
gmhA
mutant were used in the temperature-dependent rabbit model for dermal lesion production by
H. ducreyi
, the
gmhA
mutant was found to be substantially less virulent than the wild-type parent strain. The
H. ducreyi gmhA
gene was amplified by PCR from the
H. ducreyi
chromosome and cloned into the pLS88 vector. When the
H. ducreyi gmhA
gene was present in
trans
in
gmhA
mutant 35000.252, expression of the
gmhA
gene product restored the virulence of this mutant to wild-type levels. These results indicate that the
gmhA
gene product of
H. ducreyi
is essential for the expression of wild-type LOS by this pathogen.
Coiling Phagocytosis of Trypanosomatids and Fungal Cells
Authors: Rittig M. G. , Schröppel K. , Seack K.-H. , Sander U. , N’Diaye E.-N. , Maridonneau-Parini I. , Solbach W. , Bogdan C.
Published: March 2020

Coiling phagocytosis has previously been studied only with the bacteria
Legionella pneumophila
and
Borrelia burgdorferi
, and the results were inconsistent. To learn more about this unconventional phagocytic mechanism, the uptake of various eukaryotic microorganisms by human monocytes, murine macrophages, and murine dendritic cells was investigated in vitro by video and electron microscopy. Unconventional phagocytosis of
Leishmania
spp. promastigotes,
Trypanosoma cruzi
trypomastigotes,
Candida albicans
hyphae, and zymosan particles from
Saccharomyces cerevisiae
differed in (i) morphology (rotating unilateral pseudopods with the trypanosomatids, overlapping bilateral pseudopods with the fungi), (ii) frequency (high with
Leishmania
; occasional with the fungi; rare with
T. cruzi
), (iii) duration (rapid with zymosan; moderate with the trypanosomatids; slow with
C. albicans
), (iv) localization along the promastigotes (flagellum of
Leishmania major
and
L. aethiopica
; flagellum or posterior pole of
L. donovani
), and (v) dependence on complement (strong with
L. major
and
L. donovani
; moderate with the fungi; none with
L. aethiopica
). All of these various types of unconventional phagocytosis gave rise to similar pseudopod stacks which eventually transformed to a regular phagosome. Further video microscopic studies with
L. major
provided evidence for a cytosolic localization, synchronized replication, and exocytic release of the parasites, extending traditional concepts about leishmanial infection of host cells. It is concluded that coiling phagocytosis comprises phenotypically similar consequences of various disturbances in conventional phagocytosis rather than representing a single separate mechanism.
An Essential Role for Interleukin-5 and Eosinophils in Helminth-Induced Airway Hyperresponsiveness
Authors: Hall Laurie R. , Mehlotra Rajeev K. , Higgins Alan W. , Haxhiu Musa A. , Pearlman Eric
Published: March 2020

Infection with the parasitic helminthBrugia malayican result in development of a severe asthmatic response termed tropical pulmonary eosinophilia. This disease, thought to result from a host inflammatory response to blood parasites which become trapped in the lung microvasculature, is characterized by a profound eosinophilic infiltration into the lungs. Recruitment of eosinophils also correlates with the development of airway hyperresponsiveness (AHR) to cholinergic agonists and severe asthmatic symptoms. Our studies examined the role of interleukin-5 (IL-5) in helminth-induced pulmonary eosinophilia and AHR. C57BL/6 mice immunized with killedB. malayimicrofilariae and challenged intravenously with live microfilariae exhibit many of the characteristics of human disease, including peripheral and pulmonary eosinophilia. Cells recovered by bronchoalveolar lavage of sensitized mice consisted of 3.8% eosinophils on day 1 postchallenge and 84% on day 10. Extracellular major basic protein was present on the surface of airway epithelial cells as early as day 1 and continued to be evident after 8 days, indicating sustained activation and degranulation of eosinophils in the lung. These histologic changes correlated with the development of AHR to carbachol. In contrast to immunocompetent mice, immunization and challenge withB. malayiin IL-5−/−mice did not induce peripheral or pulmonary eosinophilia, and these mice failed to show AHR in response to cholinergic agonists. Taken together, these data indicate that IL-5 and eosinophils are required for the induction of AHR by filarial helminths.
Induction of Fibrinogen Expression in the Lung Epithelium duringPneumocystis cariniiPneumonia
Authors: Simpson-Haidaris Patricia J. , Courtney Mary-Anne , Wright Terry W. , Goss Rachel , Harmsen Allen , Gigliotti Francis
Published: March 2020

Pneumocystis cariniiis an important pulmonary pathogen responsible for morbidity and mortality in patients with AIDS. The acute-phase response (APR), the primary mechanism used by the body to restore homeostasis following infection, is characterized by increased levels of circulating fibrinogen (FBG). Although the liver is the primary site of increased FBG synthesis during the APR, we unexpectedly discovered that FBG is synthesized and secreted by lung alveolar epithelial cells in vitro during an inflammatory stimulus. Therefore, we sought to determine whether lung epithelial cells produce FBG in vivo using animal models ofP. cariniipneumonia (PCP). Inflammation was noted by an influx of macrophages toP. carinii-infected alveoli. Northern hybridization revealed that γ-FBG mRNA increased two- to fivefold inP. carinii-infected lung tissue, while RNA in situ hybridization demonstrated increased levels of γ-FBG mRNA in the lung epithelium. Immunoelectron microscopy detected lung epithelial cell-specific production of FBG, suggesting induction of a localized inflammatory response resembling the APR. A systemic APR was confirmed by a two- to fivefold upregulation of the levels of hepatic γ-FBG mRNA in animals with PCP, resulting in a corresponding increase in levels of FBG in plasma. Furthermore, immunoelectron microscopy revealed the presence of FBG at the junction of cell membranes of trophic forms ofP. cariniiorganisms aggregated along the alveolar epithelium. These results implicate FBG in the pathogenesis of PCP in a manner similar to that of the adhesive glycoproteins fibronectin and vitronectin, which are known to participate in intra-alveolar aggregation of organisms and adherence ofP. cariniito the lung epithelium.
Actinobacillus actinomycetemcomitansLeukotoxin Induces Apoptosis in HL-60 Cells
Authors: Korostoff Jonathan , Wang Jian Fei , Kieba Irene , Miller Mark , Shenker Bruce J. , Lally Edward T.
Published: March 2020

Actinobacillus actinomycetemcomitansleukotoxin (Ltx) is a member of the repeats-in-toxin (RTX) family of pore-forming toxins and kills human immune cells. Currently, it remains unclear whether toxin-mediated killing of target cells involves the induction of necrosis or apoptosis. Therefore, the goal of this investigation was to determine whether Ltx is capable of causing apoptotic cell death in toxin-sensitive promyelocytic HL-60 cells. Multiparameter flow cytometric analysis of toxin-treated cells stained with Hoechst 33258 (or 33342) and 7-aminoactinomycin D allowed us to identify four populations: viable cells, early apoptotic cells, late apoptotic and/or secondarily necrotic cells, and a final population that was composed of cellular debris. Compared with control cells, HL-60 cells treated with Ltx exhibited a gradual decrease in forward light scatter with a coincident increase in side light scatter, indicative of a decrease in cell size and organelle condensation, respectively. Additional experiments demonstrated that Ltx-treated cells showed evidence of internucleosomal DNA fragmentation and phosphatidylserine translocation. The results of our studies clearly demonstrate that Ltx can kill HL-60 cells by inducing apoptosis. We hypothesize that elimination of acute inflammatory cells via this mechanism plays a critical role in the pathogenesis of diseases caused byA. actinomycetemeomitans.
The
Helicobacter pylori
UreI Protein Is Not Involved in Urease Activity but Is Essential for Bacterial Survival In Vivo
Authors: Skouloubris Stéphane , Thiberge Jean-Michel , Labigne Agnès , De Reuse Hilde
Published: March 2020

We produced defined isogenic
Helicobacter pylori ureI
mutants to investigate the function of UreI, the product of one of the genes of the urease cluster. The insertion of a
cat
cassette had a strong polar effect on the expression of the downstream urease genes, resulting in very weak urease activity. Urease activity, measured in vitro, was normal in a strain in which
ureI
was almost completely deleted and replaced with a nonpolar cassette. In contrast to previous reports, we thus found that the product of
ureI
was not necessary for the synthesis of active urease. Experiments with the mouse-adapted
H. pylori
SS1 strain carrying the nonpolar
ureI
deletion showed that UreI is essential for
H. pylori
survival in vivo and/or colonization of the mouse stomach. The replacement of
ureI
with the nonpolar cassette strongly reduced
H. pylori
survival in acidic conditions (1-h incubation in phosphate-buffered saline solution at pH 2.2) in the presence of 10 mM urea. UreI is predicted to be an integral membrane protein and may therefore be involved in a transport process essential for
H. pylori
survival in vivo.
Isolation of
Enterococcus faecalis
Clinical Isolates That Efficiently Adhere to Human Bladder Carcinoma T24 Cells and Inhibition of Adhesion by Fibronectin and Trypsin Treatment
Authors: Shiono Akihiko , Ike Yasuyoshi
Published: March 2020

The adherence of
Enterococcus faecalis
strains to human T24 cells was examined by scanning electron microscopy. Five highly adhesive strains were identified from 30 strains isolated from the urine of patients with urinary tract infections. No efficiently adhesive strains were found among the 30 strains isolated from the feces of healthy students. The five isolated strains also adhered efficiently to human bladder epithelial cells. Analysis of restriction endonuclease-digested plasmid DNAs and chromosome DNAs showed that the five strains were different strains isolated from different patients. The adhesiveness of these strains was inhibited by treatment with fibronectin or trypsin, implying that a specific protein (adhesin) on the bacterial cell surface mediates adherence to fibronectin on the host cell surfaces, and the adhesin differs from the reported adhesins.
In Vivo Expression and Immunoadjuvancy of a Mutant of Heat-Labile Enterotoxin ofEscherichia coliin Vaccine and Vector Strains ofVibrio cholerae
Authors: Ryan Edward T. , Crean Thomas I. , John Manohar , Butterton Joan R. , Clements John D. , Calderwood Stephen B.
Published: March 2020

Vibrio choleraesecretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) ofEscherichia coli, the latter when expressed inV. cholerae. Both toxins are also potent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed byE. colihave been developed. One such mutant LT molecule has the substitution of a glycine residue for arginine-192 [LT(R192G)]. Live attenuated strains ofV. choleraethat have been used both asV. choleraevaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed. In order to ascertain whether LT(R192G)can act as an immunoadjuvant when expressed in vivo byV. cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains ofV. cholerae, Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous antigens in the chromosome of the vaccine vectors. We found that LT(R192G)was expressed from pCS95 in vitro by bothE. coliandV. choleraestrains but that LT(R192G)was detectable in the supernatant fraction ofV. choleraecultures only. In order to assess potential immunoadjuvanticity, groups of germfree mice were inoculated with the threeV. choleraevaccine strains alone and compared to groups inoculated with theV. choleraevaccine strains supplemented with purified CT as an oral immunoadjuvant orV. choleraevaccine strains expressing LT(R192G)from pCS95. We found that mice continued to pass stool containingV. choleraestrains with pCS95 for at least 4 days after oral inoculation, the last day evaluated. We found that inoculation withV. choleraevaccine strains containing pCS95 resulted in anti-LT(R192G)immune responses, confirming in vivo expression. We were unable to detect immune responses directed against the heterologous antigens expressed at low levels in any group of animals, including animals that received purified CT as an immunoadjuvant. We were, however, able to measure increased vibriocidal immune responses against vaccine strains in animals that receivedV. choleraevaccine strains expressing LT(R192G)from pCS95 compared to the responses in animals that receivedV. choleraevaccine strains alone. These results demonstrate that mutant LT molecules can be expressed in vivo by attenuated vaccine strains ofV. choleraeand that such expression can result in an immunoadjuvant effect.
The
rgg
Gene of
Streptococcus pyogenes
NZ131 Positively Influences Extracellular SPE B Production
Authors: Chaussee Michael S. , Ajdic Dragana , Ferretti Joseph J.
Published: March 2020

Streptococcus pyogenes
produces several extracellular proteins, including streptococcal erythrogenic toxin B (SPE B), also known as streptococcal pyrogenic exotoxin B and streptococcal proteinase. Several reports suggest that SPE B contributes to the virulence associated with
S. pyogenes
; however, little is known about its regulation. Nucleotide sequence data revealed the presence, upstream of the
speB
gene, of a gene, designated
rgg
, that was predicted to encode a polypeptide similar to previously described positive regulatory factors. The putative Rgg polypeptide of
S. pyogenes
NZ131 consisted of 280 amino acids and had a predicted molecular weight of 33,246. To assess the potential role of Rgg in the production of SPE B, the
rgg
gene was insertionally inactivated in
S. pyogenes
NZ131, which resulted in markedly decreased SPE B production, as determined both by immunoblotting and caseinolytic activity on agar plates. However, the production of other extracellular products, including streptolysin O, streptokinase, and DNase, was not affected. Complementation of the
rgg
mutant with an intact
rgg
gene copy in
S. pyogenes
NZ131 could restore SPE B production and confirmed that the
rgg
gene product is involved in the production of SPE B.
Antibodies Reactive with the N-Terminal Domain ofPlasmodium falciparumSerine Repeat Antigen Inhibit Cell Proliferation by Agglutinating Merozoites and Schizonts
Authors: Pang Xin-Li , Mitamura Toshihide , Horii Toshihiro
Published: March 2020

The serine repeat antigen (SERA) is a vaccine candidate antigen ofPlasmodium falciparum. Immunization of mice withEscherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47′) induced an antiserum that was inhibitory to parasite growth in vitro. Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages. When Percoll-purified schizonts were cultured with the affinity-purified SE47′-specific antibodies, schizonts and merozoites were agglutinated. Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47′-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC). Furthermore, double-fluorescence staining against SE47′-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47′-specific-IgG-bound schizonts after cultivation. These observations suggest that the SE47′-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.
Severe Impairment in Early Host Defense againstCandida albicansin Mice Deficient in Myeloperoxidase
Authors: Aratani Yasuaki , Koyama Hideki , Nyui Sei-ichiro , Suzuki Kazuo , Kura Fumiaki , Maeda Nobuyo
Published: March 2020

Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitonealStaphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection withCandida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injectedC. albicansinto various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.
Characterization of a Novel Trypanosome Lytic Factor from Human Serum
Authors: Raper Jayne , Fung Ramie , Ghiso Jorge , Nussenzweig Victor , Tomlinson Stephen
Published: March 2020

Natural resistance of humans to the cattle pathogen
Trypanosoma brucei brucei
has been attributed to the presence in human serum of nonimmune factors that lyse the parasite. Normal human serum contains two trypanosome lytic factors (TLFs). TLF1 is a 500-kDa lipoprotein, which is reported to contain apolipoprotein A-I (apoA-I), haptoglobin-related protein (Hpr), hemoglobin, paraoxonase, and apoA-II, whereas TLF2 is a larger, poorly characterized particle. We report here a new immunoaffinity-based purification procedure for TLF2 and TLF1, as well as further characterization of the components of each purified TLF. Immunoaffinity-purified TLF1 has a specific activity 10-fold higher than that of TLF1 purified by previously described methods. Moreover, we find that TLF1 is a lipoprotein particle that contains mainly apoA-I and Hpr, trace amounts of paraoxonase, apoA-II, and haptoglobin, but no detectable hemoglobin. Characterization of TLF2 reveals that it is a 1,000-kDa protein complex containing mainly immunoglobulin M, apoA-I, and Hpr but less than 1% detectable lipid.
Immune Response to
Yersinia
Outer Proteins and Other
Yersinia pestis
Antigens after Experimental Plague Infection in Mice
Authors: Benner Gretchen E. , Andrews Gerard P. , Byrne W. Russell , Strachan Susan D. , Sample Allen K. , Heath David G. , Friedlander Arthur M.
Published: March 2020

There is limited information concerning the nature and extent of the immune response to the virulence determinants of
Yersinia pestis
during the course of plague infection. In this study, we evaluated the humoral immune response of mice that survived lethal
Y. pestis
aerosol challenge after antibiotic treatment. Such a model may replicate the clinical situation in humans and indicate which virulence determinants are expressed in vivo. Immunoglobulin G enzyme-linked immunosorbent assay and immunoblotting were performed by using purified, recombinant antigens including F1, V antigen, YpkA, YopH, YopM, YopB, YopD, YopN, YopE, YopK, plasminogen activator protease (Pla), and pH 6 antigen as well as purified lipopolysaccharide. The major antigens recognized by murine convalescent sera were F1, V antigen, YopH, YopM, YopD, and Pla. Early treatment with antibiotics tended to reduce the immune response and differences between antibiotic treatment regimens were noted. These results may indicate that only some virulence factors are expressed and/or immunogenic during infection. This information may prove useful for selecting potential vaccine candidates and for developing improved serologic diagnostic assays.
Middle Ear Fluid Cytokine and Inflammatory Cell Kinetics in the Chinchilla Otitis Media Model
Authors: Sato Katsuro , Liebeler Carol L. , Quartey Moses K. , Le Chap T. , Giebink G. Scott
Published: March 2020

Streptococcus pneumoniae
is the most frequent microbe causing middle ear infection. The pathophysiology of pneumococcal otitis media has been characterized by measurement of local inflammatory mediators such as inflammatory cells, lysozyme, oxidative metabolic products, and inflammatory cytokines. The role of cytokines in bacterial infection has been elucidated with animal models, and interleukin (IL)-1β, IL-6, and IL-8 and tumor necrosis factor alpha (TNF-α) are recognized as being important local mediators in acute inflammation. We characterized middle ear inflammatory responses in the chinchilla otitis media model after injecting a very small number of viable pneumococci into the middle ear, similar to the natural course of infection. Middle ear fluid (MEF) concentrations of IL-1β, IL-6, IL-8, and TNF-α were measured by using anti-human cytokine enzyme-linked immunosorbent assay reagents. IL-1β showed the earliest peak, at 6 h after inoculation, whereas IL-6, IL-8, and TNF-α concentrations were increasing 72 h after pneumococcal inoculation. IL-6, IL-8, and TNF-α but not IL-1β concentrations correlated significantly with total inflammatory cell numbers in MEF, and all four cytokines correlated significantly with MEF neutrophil concentration. Several intercytokine correlations were significant. Cytokines, therefore, participate in the early middle ear inflammatory response to
S. pneumoniae.
Integration Host Factor and Cyclic AMP Receptor Protein Are Required for TyrR-Mediated Activation of
tpl
in
Citrobacter freundii
Authors: Bai Qing , Somerville Ronald L.
Published: March 2020

The
tpl
gene of
Citrobacter freundii
encodes an enzyme that catalyzes the conversion of
l
-tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates −272.5, −158.5, and −49.5, respectively, were identified in the upstream region of the
tpl
promoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation of
tpl
required integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate −85 and that there are CRP binding sites centered at coordinates −220 and −250. Mutational alteration of the IHF binding site reduced the efficiency of the
tpl
promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into
tpl
promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased when
tpl
DNA was present. The participation of both IHF and CRP in the activation of the
tpl
promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of the
tpl
promoter is presented.
Role of SarA in Virulence Determinant Production and Environmental Signal Transduction in
Staphylococcus aureus
Authors: Chan Pan F. , Foster Simon J.
Published: March 2020

The staphylococcal accessory regulator (encoded by
sarA
) is an important global regulator of virulence factor biosynthesis in
Staphylococcus aureus
. To further characterize its role in virulence determinant production, an
sarA
knockout mutant was created by insertion of a kanamycin antibiotic resistance cassette into the
sarA
gene. N-terminal sequencing of exoproteins down-regulated by
sarA
identified several putative proteases, including a V8 serine protease and a novel metalloprotease, as the major extracellular proteins repressed by
sarA
. In kinetic studies, the
sarA
mutation delays the onset of α-hemolysin (encoded by
hla
) expression and reduces levels of
hla
to approximately 40% of the parent strain level. Furthermore, SarA plays a role in signal transduction in response to microaerobic growth since levels of
hla
were much lower in a microaerobic environment than after aerobic growth in the
sarA
mutant. An exoprotein exhibiting hemolysin activity on sheep blood, and up-regulated by
sarA
independently of the accessory gene regulator (encoded by
agr
), was specifically induced microaerobically. Transcriptional gene fusion and Western analysis revealed that
sarA
up-regulates both toxic shock syndrome toxin 1 gene (
tst
) expression and staphylococcal enterotoxin B production, respectively. This study demonstrates the role of
sarA
as a signal transduction regulatory component in response to aeration stimuli and suggests that
sarA
functions as a major repressor of protease activity. The possible role of proteases as regulators of virulence determinant stability is discussed.
Corepression of the P1 Addiction Operon by Phd and Doc
Authors: Magnuson Roy , Yarmolinsky Michael B.
Published: March 2020

The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation,
docH66Y
. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.
Fourier Transform Infrared Spectroscopy Is a New Option for Outbreak Investigation: a Retrospective Analysis of an Extended-Spectrum-Beta-Lactamase-Producing Klebsiella pneumoniae Outbreak in a Neonatal Intensive Care Unit
Authors: Rakovitsky Nadya , Frenk Sammy , Kon Hadas , Schwartz David , Temkin Elizabeth , Solter Ester , Paikin Svetlana , Cohen Regev , Schwaber Mitchell J. , Carmeli Yehuda , Lellouche Jonathan
Published: March 2020

The IR Biotyper is a new automated typing system based on Fourier-transform infrared (FT-IR) spectroscopy that gives results within 4 h. We aimed (i) to use the IR Biotyper to retrospectively analyze an outbreak of extended-spectrum beta-lactamase-producing
Klebsiella pneumoniae
(ESBL-KP) in a neonatal intensive care unit and to compare results to BOX-PCR and whole-genome sequencing (WGS) results as the gold standard and (ii) to assess how the cutoff values used to define clusters affect the discriminatory power of the IR Biotyper.
An Analytical Model Applied to a Multicenter Pneumococcal Enzyme-Linked Immunosorbent Assay Study
Authors: Plikaytis Brian D. , Goldblatt David , Frasch Carl E. , Blondeau Christine , Bybel Michael J. , Giebink G. Scott , Jonsdottir Ingileif , Käyhty Helena , Konradsen Helle Bossen , Madore Dace V. , Nahm Moon H. , Schulman Cheryl A. , Holder Patricia F. , Lezhava Tamar , Elie Cheryl M. , Carlone George M.
Published: March 2020

Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.
PCR and Restriction Endonuclease Analysis for Rapid Identification of Human Adenovirus Subgenera
Authors: Elnifro Elfath M. , Cooper Robert J. , Klapper Paul E. , Bailey Andrew S.
Published: March 2020

Subgenus identification of adenoviruses is of clinical importance and is as informative as identification by serotype in most clinical situations. A PCR-based identification of adenovirus subgenera A, B, C, D, E, and F and sometimes serotypes is described. The PCR uses nonnested primer pair ADRJC1-ADRJC2, which targets a highly conserved region of the adenovirus hexon gene, has a sensitivity of 10 to 40 copies of adenovirus type 2 (Ad2) DNA, and generates 140-bp PCR products from adenovirus serotypes representative of all the subgroups. The PCR products of all subgroups can be differentiated on the basis of the restriction fragment patterns produced by a total of five restriction endonucleases. In addition, serotypes Ad40 and Ad41 (subgroup F) and important serotypes of subgroup D (Ad8, Ad10, Ad19, and Ad37) can easily be differentiated, but serotypes within subgroups B and C cannot. The method was assessed by blind subgenus identification of 56 miscellaneous clinical isolates of adenoviruses. The identities of these isolates at the subgenus level by the PCR correlated 91% (51 of 56) with the results of serotyping by the neutralization test, and 9% (5 of 56) of clinical isolates produced discordant results.