bluuebunny/crossref_metadata_2025 huggingface dataset:
Lymphocytes Tγδ in clinically normal skin and peripheral blood of patients with systemic lupus erythematosus and their correlation with disease activity
Authors: Robak Ewa , Niewiadomska Hanna , Robak Tadeusz , Bartkowiak Jacek , Bloński Jerzy Z. , Woźniacka Anna , Pomorski Lech , Sysa-Jędrzejowska Anna
Published: November 2003

Human Tγσ lymphocytes constitute from 1 to 15% of all peripheral blood lymphocytes. Recent work has demonstrated that this population plays a major role in the pathogenesis of infectious and immune diseases. Increased numbers of γσ T cells have been found in affected skin from systemic sclerosis and chronic cutaneous lupus erythematosus patients.

In our study, we have determined the numbers of Tγσ lymphocytes and their subpopulations in peripheral blood from 29 patients with systemic lupus erythematosus (SLE) and in 19 healthy volunteers using flow cytometry and specific monoclonal antibodies. The same cells in uninvolved skin from SLE patients and human controls using immunohistochemical analysis were estimated. T‐Cell receptor (TCR) delta chain gene rearrangement was identified with primers for Vσ1, Vσ2 and Vσ3 by the polymerase chain reaction. Statistical analysis showed a significantly decreased number of γσ T cells in SLE patients (26.4 Ī 16.9/μl) compared with the control group (55.3 Ī 20.6/μl) (p < 0.001). The number of Vσ2 TCR+ and Vγ9 TCR+ subpopulations was also lower in SLE patients than in healthy persons. No statistical correlation between disease activity and the number of γσ T cells was demonstrated. The percentage of T γσ lymphocytes in clinically normal skin from SLE patients was twice (22.0 Ī 9.4%) that found in the skin from healthy persons (11.1 Ī 5.5%) (p < 0.002). Higher percentages of the Vσ2 TCR+ and Vγ9 TCR+ subpopulation of lymphocytes were found in the skin from SLE patients. We have also found positive correlation between the percentage of Tγσ lymphocytes in skin and the activity of SLE (r = 0.594,  p < 0.001), and between subpopulation Vσ3 TCR+ and disease activity
(r = 0.659,  p < 0.001). In conclusion, the results of our studies demonstrate that, in patients with SLE, accumulation of Tγσ lymphocytes can be seen in clinically normal skin, and the percentage of these cells correlates with the activity of the disease.
Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions
Authors: Cardoso Diva F. , Lopes-Ferreira Monica , Faquim-Mauro Eliana L. , Macedo Mahasti S. , Farsky Sandra H. P.
Published: November 2003

Background: Crotoxin (CTX) is a potent neurotoxin from
Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions.

Aim: The aim of this paper was to investigate the mechanisms involved in these impaired responses.

Materials and methods: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)‐6, IL‐10, interferon (IFN)‐γ and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL‐4, IL6, IL‐10 or IFN‐γ secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment.

Results: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSAtreated animals. Increased levels of IL‐6, IL‐10 and corticosterone were also detected in CTX‐injected animals. IFN‐ γ levels were not modified after treatments. In contrast, spleen cells obtained from CTX‐treated animals and stimulated with concanavalin A secreted less IL‐10 and IL‐4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration.

Conclusions: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV.CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.
Evidence for local inflammation in complex regional pain syndrome type 1
Authors: Huygen Frank J. P. M. , de Bruijn Anke G. J. , de Bruin Martha T. , Groeneweg J. George , Klein Jan , Zijlstra Freek J.
Published: November 2003

Background: The pathophysiology of complex regional pain syndrome type 1 (CRPS 1) is still a matter of debate. Peripheral afferent, efferent and central mechanisms are supposed. Based on clinical signs and symptoms (e.g. oedema, local temperature changes and chronic pain) local inflammation is suspected.

Aim: To determine the involvement of neuropetides, cytokines and eicosanoids as locally formed mediators of inflammation.

Methods: In this study, nine patients with proven CRPS 1 were included. Disease activity and impairment was determined by means of a Visual Analogue Scale, the McGill Pain Questionnaire, the difference in volume and temperature between involved and uninvolved extremities, and the reduction in active range of motion of the involved extremity. Venous blood was sampled from and suction blisters made on the involved and uninvolved extremities for measurement of cytokines interleukin (IL)‐6, IL‐1β and tumour necrosis factor‐α (TNF‐α), the neuropetides NPY and CRGP, and prostaglandin E2.

Results: The patients included in this study did have a moderate to serious disease activity and impairment. In plasma, no changes of mediators of inflammation were observed. In blister fluid, however, significantly higher levels of IL‐6 and TNF‐α in the involved extremity were observed in comparison with the uninvolved extremity.

Conclusions: This is the first time that involvement of mediators of inflammation in CRPS 1 has been so clearly and directly demonstrated. This observation opens new approaches for the succesful use and development of immunosuppressives in CRPS 1.
Cytokine profile after rubella vaccine inoculation: evidence of the immunosuppressive effect of vaccination
Authors: Pukhalsky Alexander L. , Shmarina Galina V. , Bliacher Maria S. , Fedorova Irina M. , Toptygina Anna P. , Fisenko Julia J. , Alioshkin Vladimir A.
Published: November 2003

Background: Immunization with live virus vaccines may cause an immunosuppression with lymphopaenia, impaired cytokine production and defective lymphocyte response to mitogenes. These abnormalities were described in subjects vaccinated against measles. This study was performed to analyse the host immune response related to immunosuppression in subjects vaccinated with live attenuated rubella vaccine.

Methods: Eighteen schoolgirls, aged 11‐13 years, were vaccinated with live attenuated rubella vaccine Rudivax®. Before immunization, and 7 and 30 days after, peripheral blood was collected. Cellular fractions were subjected to flow cytometric analysis, and the lymphocyte response to phytohaemagglutinin was investigated. Plasma samples were analysed for cytokines (interleukin (IL)‐4, IL‐10, tumour necrosis factor‐α, and interferon‐γ) by enzyme‐linked immunosorbent assay techniques.

Results: On day 7 after vaccination, the number of each lymphocyte subset was decreased; however, only for CD3 and CD4 lymphocytes has a significant reduction been shown. On the contrary, tumour necrosis factor‐α and IL‐10 levels markedly increased and amounted to its maximum on day 30. Simultaneously, a significant reduction in plasma interferon‐γ and a profound decrease of the lymphocyte response to phytohaemagglutinin were shown. The changes were accompanied with marked elevation of plasma IL‐4.

Conclusions: Our data indicate that the vaccination with live attenuated rubella vaccine results in moderate but sustained immune disturbance. The signs of immunosuppression, including defective lymphocyte response to mitogene and impaired cytokine production, may persist for at least 1 month after vaccination.
Hydroxyl‐platelet‐activating factor exists in blood of healthy volunteers and periodontal patients
Authors: Antonopoulou Smaragdi , Tsoupras Alexandros , Baltas George , Kotsifaki Helen , Mantzavinos Zacharias , Demopoulos Constantinos A.
Published: November 2003

Periodontal diseases are localized chronic inflammatory conditions of the gingival and underlying bone and connective tissue. Platelet‐activating factor (PAF), a potent inflammatory phospholipid mediator that has been previously detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid and in saliva, is implicated in periodontal disease. Our results from previous studies showed that the biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl‐PAF analogue. In this study, hydroxyl‐PAF analogue was detected for the first time in human blood derived from patients with chronic periodontitis as well as from periodontally healthy volunteers. The hydroxyl‐PAF analogue was purified by high‐performance liquid chromatography, detected by biological assays and identified by electrospray analysis. In addition, the quantitative determination of PAF and hydroxyl‐PAF analogue (expressed as PAF‐like activity) showed a statistically significant increase in the ratio of hydroxyl‐PAF analogue levels to PAF levels in periodontal patients, suggesting that this bioactive lipid may play a role in oral inflammation.
On stochastic inequalities and comparisons of reliabilitymeasures for weighted distributions
Authors: Oluyede Broderick O. , George E. Olusegun
Published: November 2003

Inequalities, relations and stochastic orderings, as well as useful ageing notions for weighted distributions are established. Also presented are preservation and stability results and comparisons for weighted and length‐biased distributions. Relations for length‐biased and equilibrium distributions as examples of weighted distributions are also presented.
QCA implementation of a multichannel filter for image processing
Authors: Cardenas-Barrera J. L. , Plataniotis K. N. , Venetsanopoulos A. N.
Published: November 2003

A novel way to implement a color image filter is introduced here. The quantum‐dot cellular automata (QCA) framework is adopted due to its adequacy to implement highly parallel systems. The principle behind the new design is explained in detail, and simulation results are included to demonstrate the effectiveness of the proposed method.
Linear discrete‐time systems with Markovian jumps and modedependent time‐delay: Stability and stabilizability
Authors: Boukas E. K. , Shi P. , Karan M. , Kaya C. Y.
Published: November 2003

This paper considers stochastic stability and stochastic stabilizability of linear discrete‐time systems with Markovian jumps and mode‐dependent time‐delays. Linear matrix inequality (LMI) techniques are used to obtain sufficient conditions for the stochastic stability and stochastic stabilizability of this class of systems. A control design algorithm is also provided. A numerical example is given to demonstrate the effectiveness of the obtained theoretical results.
A new formulation of the equivalent thermal in optimization ofhydrothermal systems
Authors: Bayón L. , Grau J. M. , Suárez P.
Published: November 2003

In this paper, we revise the classical formulation of the problem depriving it of the concepts that are superfluous from the mathematical point of view. We observe that a number of power stations can be substituted by a single one that behaves equivalently to the entire set. Proceeding in this way, we obtain a variational formulation in its purest sense (without restrictions). This formulation allows us to employ the theory of calculus of variations to the highest degree. We then calculate the equivalent minimizer in the case where the cost functions are second‐order polynomials. We prove that the equivalent minimizer is a second‐order polynomial with piece‐wise
constant coefficients. Moreover, it belongs to the class C1. Finally, we present various examples prompted by real systems and perform the proposed algorithms using Mathematica.
DSCR1(Adapt78)‐‐A Janus Gene Providing Stress Protection but Causing Alzheimer's Disease?
Authors: Ermak Gennady , Davies Kelvin
Published: November 2003

Alzheimer's disease is associated with the formation of paired helical filaments composed of hyperphospharylated tau protein. Phosphatase 2B, calcineurin can dephosphorylate the tau protein and, therefore, might prevent the assembly of paired helical filaments and even Alzheimer's disease. Calcipressin 1, the DSCR1(Adapt78) gene product, can bind and inactivate calcineurin. Here we hypothesize that while short‐term induction of calcipressin1 can provide stress protection, its long‐term or chronic induction may cause gradual accumulation of hyperphosphorylated tau protein, eventually leading to Alzheimer's disease. IUBMB Life, 55: 29‐31, 2003
Herpes Virus Replication
Authors: Boehmer Paul , Nimonkar Amitabh
Published: November 2003

Herpesviruses are large double stranded DNA animal viruses with the distinguishing ability to establish latent, life‐long infections. To date, eight human herpesviruses that exhibit distinct biological and corresponding pathological/clinical properties have been identified. During their life cycles, herpesviruses execute an intricate chain of events geared towards optimizing their replication. This sets an interesting paradigm to study fundamental biological processes. This review summarizes recent developments in herpesvirus research with emphasis on genome transactions, particularly with respect to the prototypic herpes simplex virus type‐1. IUBMB Life, 55: 13‐22, 2003
Does Palmitoylation Target Estrogen Receptors to Plasma Membrane Caveolae?
Authors: Acconcia Filippo , Bocedi Alessio , Ascenzi Paolo , Marino Maria
Published: November 2003

The human nuclear estrogen receptors (i.e., ERα and ERβ), as other transcriptional factors, regulate cellular processes inducing genomic events. In addition, the binding of 17β‐estradiol to ERs induces different membrane initiating non‐genomic signaling. The non‐genomic effects of ER are independent of transcriptional activity of the receptor and have been attributed to membrane ERs belonging to a signaling complex localized in caveolae. Here, we postulate that S‐acylation of cysteine residue(s) present in the ligand binding domain of ERα and ERβ may play a critical role in the membrane caveolar localization of the receptor and in the formation of the 'steroid signalosome'. IUBMB Life, 55: 33‐35, 2003
Biphasic Role of Platelet‐Activating Factor in Oral Mucosal Ulcer Healing
Authors: Slomiany Bronislaw , Slomiany Amalia
Published: November 2003

Platelet‐activating factor (PAF) is a potent phospholipid‐derived messenger molecule involved in a number of pathological conditions, including mediation of inflammatory cascades associated with wound healing. We investigated prophylactic and therapeutic effects of a specific PAF antagonist, BN52020, on the course of experimentally induced oral mucosal ulcer healing. The prophylactic BN52020 administration produced an accelerated ulcer healing that was characterized by a marked induction in COX‐2 enzyme protein expression and the substantial decline in apoptosis, TNF‐ α, and NOS‐2 activity. A delay in ulcer healing, however, occurred with the therapeutic BN52020 administration, and this effect of the agent was reflected in a decreased expression of COX‐2 protein, higher rate of apoptosis, and the elevated level of TNF‐ αand NOS‐2. Our findings implicate PAF requirement in orderly progression of the events involved in oral tissue repair, and suggest that the interference with its actions during healing process results in the suppression of COX‐2‐derived anti‐inflammatory prostaglandins that delay the mucosal repair. IUBMB Life, 55: 483‐490, 2003
Rotary Movements within the ATP Synthase do not Constitute an Obligatory Element of the Catalytic Mechanism
Authors: Berden Jan
Published: November 2003

After a brief history of the proposals for the mechanism of the ATP synthase, the main experimental arguments for a rotational mechanism of catalysis are analyzed and on the basis of this analysis it is concluded that no evidence has been provided for rotation as an obligatory element of the catalytic mechanism. On the other hand, the experimental evidence in favor of a two‐sites catalytic mechanism, derived from various approaches and not compatible with a three‐sites rotary mechanism, appear to be very solid. Finally a brief characterization of the various nucleotide binding sites is provided and a suggestion is made how the enzyme has evolutionarily developed from a rotating machine into an asymmetrical device for energy conservation. IUBMB Life, 55: 473‐481, 2003
DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells
Authors: Cuconati Andrea , Mukherjee Chandreyee , Perez Denise , White Eileen
Published: November 2003

Expression of adenovirus E1A deregulates cell proliferation to facilitate viral DNA replication, prompting the initiation of apoptosis signaled primarily through proapoptotic BAK in productively infected cells. We demonstrate here that in uninfected cells, BAK is complexed with the anti-apoptotic BCL-2 family member Myeloid Cell Leukemia 1 (MCL-1). E1A expression during infection resulted in the specific down-regulation of MCL-1 through destabilization of the protein and loss of the mRNA. Upon loss of the MCL-1-BAK complex, BAK complexed with either BAX in proapoptotic E1B mutant adenovirus-infected cells, or with the adenovirus BCL-2 homolog E1B 19K in cells infected with the wild-type virus in which apoptosis is inhibited. Loss of MCL-1 was required to initiate the apoptotic pathway in infected cells as restoration of MCL-1 expression rescued infected cells from E1A-induced apoptosis. Analogous to E1A expression, DNA damage down-regulates MCL-1, and adenovirus infection resulted in the accumulation of phosphorylated H2AX and ataxia-telangiectasia mutant protein (ATM), hallmarks of DNA double-strand breaks. Thus, MCL-1 may function by maintaining BAK in an inactive state, and the loss of MCL-1 upon activation of the DNA damage response, perhaps through replication stress induced in virus infected cells, may be required to initiate the apoptotic response.
Marketing intelligence and account planning: insights from the experts
Authors: Crosier Keith , Pickton David
Published: November 2003

Account planning as a discipline has been somewhat obscured from academic view. In practice, it has played a significant role in the development of the marketing communications (especially, advertising) industry although it has been adopted in varying forms and with differing emphases. It has been misunderstood by many. This paper offers a summary of the insights gained from the papers contained in this special issue of Marketing Intelligence & Planning; papers that represent many years of experience in the field; papers that are a mix of academic and practitioner perspectives. Collectively, they describe this faintly mysterious discipline more completely than any other published source of which we know. What is clear from the papers is the absence of any agreed succinct description or definition of what account planning should be in the current and anticipated future marketing communications environment. This paper attempts to remedy this situation by proposing a definition of account planning derived from this collective work which others may wish to accept, develop, repudiate or (best of all) debate.
In vivo temporal and spatial distribution of depolarization and repolarization and the illusive murine T wave
Authors: Liu Gang , Iden Jason B. , Kovithavongs Kay , Gulamhusein Rashida , Duff Henry J. , Kavanagh Katherine M.
Published: November 2003

This study assessed in vivo temporal and spatial electrophysiological properties of murine hearts and the effect of manipulation of transmural action potential durations (APDs) on T wave morphology. Monophasic action potentials (MAPs) were acquired from multiple left ventricular sites. All MAPs exhibited a plateau phase, with a spike and dome appearance being present in epicardial recordings. Activation occurred from endocardial apex to epicardial apex and apex to base while repolarization occurred from base (shortest 90 η0 level of repolarization (MAP90), 95.4 ± 8.9 ms) to apex and epicardium to endocardium (longest MAP90, 110.77 ± 10.6 ms). The peak of phase 0 of the epicardial base MAP correlated with the return to baseline of the initial and usually dominant waveform of the QRS and the onset of the second usually smaller wave, which clearly occurred in early repolarization, thus establishing where depolarization ended and repolarization began on the murine ECG. This second waveform was similar to the J wave seen in larger animals. Despite temporal and spatial electrophysiological similarities, a T wave is frequently not seen on a murine ECG. There are several determinants of T wave morphology, including transmural activation time, slope of phase 3 repolarization and differences in epicardial, endocardial and M cell APDs. Experimental manipulation of murine transmural gradients by shortening epicardial MAP90 to 84% of endocardial MAP90 the epicardial/endocardial ratio in larger mammals when a positive T wave is present, resulted in a positive murine T wave. Thus, manipulation of the transmural gradients such that they are similar to larger mammals can result in T waves with similar morphology.
Repetitive firing of rat cerebellar parallel fibres after a single stimulation
Authors: Isope Philippe , Franconville Romain , Barbour Boris , Ascher Philippe
Published: November 2003

The excitatory postsynaptic currents (EPSCs) evoked in Purkinje cells (PCs) by stimulating parallel fibres (PFs) usually show a single peak, but EPSCs with multiple peaks (polyphasic EPSCs) can be observed in slices from animals older than 15 days. The EPSCs remain polyphasic when the postsynaptic current is reduced (either by reducing the intensity of the PF stimulation or by adding AMPA receptor antagonists) and when the PC membrane potential is made positive. Thus the late peaks are not due to postsynaptic active currents generated in the imperfectly clamped PC, and must arise from repetitive action potentials in the PF. Extracellular recordings from granule cell (GC) somata showed that a single PF stimulation can elicit a doublet or a train of action potentials. Both the late action potentials recorded in the GCs and the late peaks of the polyphasic EPSCs recorded in the PCs were reduced or abolished by paired‐pulse stimulation of the PF or by bath application of the GABAA agonist muscimol. The late action potentials in the GCs were also suppressed by local application of muscimol around the cell body. We propose that after a single stimulation of a PF, the antidromic invasion of the ascending axon and the granule cell can trigger a doublet or a burst of action potentials which back‐propagate into the PF (except for the first, which finds the PF still in its refractory period). The repetitive activation of the PF by a single stimulation could play a role in the induction of long‐term depression.
Functional markers and the ‘homogeneity’ of human mesenchymal stem cells
Authors: Boheler Kenneth R.
Published: November 2003

Stem cells are characterized by the abilities to self‐renew, generate large numbers of progeny and differentiate into at least one mature cell type. Bone marrow serves as a reservoir for several classes of adult stem cells. In addition to haematopoietic stem cells (HSCs), which can reconstitute the haematopoietic system of a myeloablated host, bone marrow contains a diverse population of marrow stromal cells (Herzog et al. 2003). Included among these are mesenchymal stem cells (MSCs), which ex vivo can be isolated as a relatively homogeneous and undifferentiated cell population that produces multiple mature cell types including fat, bone and cartilage (Pittenger et al. 1999). Under appropriate, but ill‐defined conditions, human MSCs (hMSCs) can also differentiate into other cell types, including excitable cells with neuronal‐, myogenic and cardiomyogenic‐like phenotypes. Because MSCs are multipotent and readily expandable in vitro, these cells have already been employed in early clinical studies, including the treatment of human myocardial infarction. Transplantation of autologous or allogeneic MSCs therefore represents a novel form of cellular therapy, which shows substantial promise for the treatment of a number of human diseases.
Oxygen Regulates Invasiveness and Virulence of Group B
Streptococcus
Authors: Johri Atul K. , Padilla Joahnna , Malin Gennady , Paoletti Lawrence C.
Published: November 2003

The facultative anaerobe group B
Streptococcus
(GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5- to 400-fold higher numbers when cultured at a cell mass doubling time (

t
d

) of 1.8 h than at a slower

t
d

of 11 h. Invasion was optimal when GBS was cultured at a

t
d

of 1.8 h in the presence of ≥5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (

t
d

of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (

t
d

of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.
TheListeria monocytogenes lemAGene Product Is Not Required forIntracellular Infection or To Activate fMIGWII-Specific TCells
Authors: D'Orazio Sarah E. F. , Velasquez Marisela , Roan Nadia R. , Naveiras-Torres Olaia , Starnbach Michael N.
Published: November 2003

Clearance of the intracellular bacterial pathogenListeria monocytogenesrequires antigen-specific CD8+T cells. Recently it was shown that activation of class Ib major histocompatibility complex (MHC)-restricted CD8+T cells alone is sufficient for immune protection against listeriae. A major component of the class Ib MHC-restricted T-cell response is T cells that recognize formylated peptide antigens presented by M3 molecules. Although threeN-formylated peptides derived fromL. monocytogenesare known to bind to M3 molecules, fMIGWII is the immunodominant epitope presented by M3 during infection of mice. The source of fMIGWII peptide is theL. monocytogenes lemAgene, which encodes a 30-kDa protein of unknown function. In this report, we describe the generation of twoL. monocytogenes lemAdeletion mutants. We show thatlemAis not required for growth of listeriae in tissue culture cells or for virulence during infection of mice. Surprisingly, we found that fMIGWII-specific T cells were still primed following infection withlemAmutant listeriae, suggesting thatL. monocytogenescontains at least one additional antigen that is cross-reactive with the fMIGWII epitope. This cross-reactive antigen appears to be a small protease-resistant molecule that is secreted byL. monocytogenes.
Divergent Interactions ofEhrlichiachaffeensis-andAnaplasma phagocytophilum-InfectedLeukocytes with Endothelial CellBarriers
Authors: Park Jinho , Choi Kyoung-Seong , Grab Dennis J. , Dumler J. Stephen
Published: November 2003

Human anaplasmosis (formerly human granulocytic ehrlichiosis) and human monocytic ehrlichiosis (HME) are emerging tick-borne infections caused by obligate intracellular bacteria in the familyAnaplasmataceae. Clinical findings include fever, headache, myalgia, leukopenia, thrombocytopenia, and hepatic inflammatory injury. WhereasEhrlichia chaffeensis(HME) often causes meningoencephalitis, this is rare withAnaplasma phagocytophiluminfection. The abilities of infected primary host monocytes and neutrophils and of infected HL-60 cells to cross human umbilical vein endothelial cell-derived EA.hy926 cell barriers and human brain microvascular cells (BMEC), a human blood-brain barrier model, were studied. Uninfected monocyte/macrophages crossed endothelial cell barriers six times more efficiently than neutrophils. MoreE. chaffeensis-infected monocytes transmigrated than uninfected monocytes, whereasA. phagocytophilumsuppressed neutrophil transmigration. Differences were not due to barrier dysfunction, as transendothelial cell resistivities were the same for uninfected cell controls. Similar results were obtained for HL-60 cells used as hosts forE. chaffeensisandA. phagocytophilum. Differential transmigration ofE. chaffeensis-andA. phagocytophilum-infected leukocytes and HL-60 cells confirmed a role for the pathogen in modifying cell migratory capacity. These results support the hypothesis thatAnaplasmataceaeintracellular infections lead to unique pathogen-specific host cell functional alterations that are likely important for pathogen survival, pathogenesis, and disease induction.
Biochemical and Immunological Characterization of Bacterially Expressed and RefoldedPlasmodium falciparum42-Kilodalton C-Terminal Merozoite Surface Protein 1
Authors: Singh Sanjay , Kennedy Michael C. , Long Carole A. , Saul Allan J. , Miller Louis H. , Stowers Anthony W.
Published: November 2003

Protection againstPlasmodium falciparumcan be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP142) inEscherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refoldedE. coliMSP142was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refoldedE. coliMSP142and tested in vitro in aP. falciparumgrowth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms ofP. falciparumMSP142. However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP119). Vaccination of nonhuman primates withE. coliMSP142in Freund's adjuvant protected six of sevenAotusmonkeys from virulent infection withP. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct,E. coliMSP142, is a viable candidate for human vaccine trials.
Identification of Dimethyl Sulfoxide Reductase inActinobacillus pleuropneumoniaeand Its Role in Infection
Authors: Baltes Nina , Hennig-Pauka Isabel , Jacobsen Ilse , Gruber Achim D. , Gerlach Gerald F.
Published: November 2003

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in anA. pleuropneumoniaeserotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein ofHaemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinishedA. pleuropneumoniaegenome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of thedmsAORF. In order to investigate the role of theA. pleuropneumoniaeDmsA protein in virulence, an isogenic deletion mutant,A. pleuropneumoniaeΔdmsA, was constructed and examined in an aerosol infection model.A. pleuropneumoniaeΔdmsAwas attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly toA. pleuropneumoniaevirulence.
An Enzymatically Active A Domain Is Required for Cholera-Like Enterotoxins To Induce a Long-Lived Blockade on the Induction of Oral Tolerance: New Method for Screening Mucosal Adjuvants
Authors: Bagley Kenneth C. , Abdelwahab Sayed F. , Tuskan Robert G. , Lewis George K.
Published: November 2003

The cholera-like enterotoxins (CLETS), cholera toxin (CT) andEscherichia coliheat-labile toxin (LT), are powerful mucosal adjuvants. Here we show that these toxins also induce a long-lived blockade (of at least 6 months) on the induction of oral tolerance when they are coadministered with the antigen ovalbumin. Strikingly, only enzymatically active CLETS induced this blockade on the induction of oral tolerance. In this regard, the enzymatically inactive mutants of CT and LT, CTK63 and LTK63, and their recombinant B pentamers, rCTB and rLTB, failed to block the induction of oral tolerance, demonstrating a stringent requirement for an enzymatically active A domain in this phenomenon. Together with the results of other recent studies, these results indicate that the enzymatic activity of CLETS, most likely cyclic AMP elevation, is responsible for their adjuvant effects. The results of this study also indicate that measuring the ability of putative mucosal adjuvants to block the induction of oral tolerance may be a superior method for measuring mucosal adjuvanticity.
Subtractive Hybridization Identifies a Novel Predicted Protein Mediating Epithelial Cell Invasion by Virulent Serotype III Group B
Streptococcus agalactiae
Authors: Adderson Elisabeth E. , Takahashi Shinji , Wang Yan , Armstrong Jianling , Miller Dylan V. , Bohnsack John F.
Published: November 2003

Group B
Streptococcus agalactiae
bacteria (group B streptococci [GBS]) are the most common cause of serious bacterial infection in newborn infants. The majority of serotype III-related cases of neonatal disease are caused by a genetically related subgroup of bacteria, restriction fragment digest pattern (RDP) type III-3, suggesting that these strains possess unique genes contributing to virulence. We used genomic subtractive hybridization to identify regions of genomic DNA unique to virulent RDP type III-3 GBS strains. Within one of these III-3-specific regions is a 1,506-bp open reading frame,
spb1
(surface protein of group B streptococcus 1). A mutant type III GBS strain lacking Spb1 was constructed in virulent RDP type III-3 strain 874391, and the interactions of the wild-type and
spb1
isogenic mutant with a variety of epithelial cells important to GBS colonization and infection were compared. While adherence of the
spb1
isogenic mutant to A549 respiratory, C2Bbe1 colonic, and HeLa cervical epithelial cells was slightly lower than that of the 874391 strain, invasion of the Spb1
−
mutant was significantly reduced with these cell lines compared to what was seen with 874391. The defect in epithelial invasion was corrected by supplying
spb1
in
trans
. These observations suggest that Spb1 contributes to the pathogenesis of neonatal GBS infection by mediating internalization of virulent serotype III GBS and confirm that understanding of the population structure of bacteria may lead to insights into the pathogenesis of human infections.
Characterization of a Protective Monoclonal AntibodyRecognizing
Staphylococcus aureus
MSCRAMM ProteinClumping FactorA
Authors: Hall Andrea E. , Domanski Paul J. , Patel Pratiksha R. , Vernachio John H. , Syribeys Peter J. , Gorovits Elena L. , Johnson Michael A. , Ross Julia M. , Hutchins Jeff T. , Patti Joseph M.
Published: November 2003

The
Staphylococcus aureus
MSCRAMM (microbial surface
components recognizing adhesive matrix molecules) protein clumping
factor A (ClfA) has been shown to be a critical virulence
factor in several experimental models of infection. This report
describes the generation, characterization, and in vivo evaluation of a
murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis
revealed that MAb 12-9 recognized ClfA protein expressed by all of the
clinical
S. aureus
strains obtained from a variety of
sources. In assays measuring whole-cell
S. aureus
binding to human fibrinogen, MAb 12-9 inhibited
S
.
aureus
binding by over 90% and displaced up to
35% of the previously adherent
S. aureus
bacteria. Furthermore, a single infusion of MAb 12-9 was protective
against an intravenous challenge with a methicillin-resistant strain of
S. aureus
in a murine sepsis model (
P
< 0.0001). These data suggest that anti-ClfA MAb 12-9 should be
further investigated as a novel immunotherapy for the treatment and
prevention of life-threatening
S. aureus
infections.
HumanAntibodies Specific for the High-Molecular-Weight Adhesion Proteins ofNontypeable
Haemophilus influenzae
Mediate OpsonophagocyticActivity
Authors: Winter Linda E. , Barenkamp Stephen J.
Published: November 2003

The
HMW1- and HMW2-like adhesion proteins of nontypeable
Haemophilus
influenzae
are expressed by 75% of these strains, and
antibodies directed against these proteins are protective in animal
models of infection. The purpose of the present study was to define the
functional activity of human antibodies specific for these proteins in
an in vitro complement-dependent opsonophagocytic assay. Human
promyelocytic cell line HL-60 served as the source of phagocytic cells,
and a commercial preparation of intravenous immunoglobulin (IVIG)
served as the source of human antibodies. High-molecular-weight (HMW)
proteins were purified from four prototype nontypeable
H.
influenzae
strains and used to prepare solid-phase affinity
columns. IVIG was adsorbed on each column to remove strain-specific
anti-HMW antibodies and to allow recovery of affinity-purified anti-HMW
antibody fractions. Unadsorbed IVIG killed each of the prototype
strains at titers of 1:80 to 1:320. HMW-adsorbed sera demonstrated
fourfold decreases in opsonophagocytic titer against the homologous
strains compared to unadsorbed IVIG. Affinity-purified anti-HMW
antibody preparations demonstrated opsonophagocytic titers of 1:20 to
1:80 against the respective homologous strains and opsonophagocytic
titers as high as 1:80 against heterologous strains. None of the
affinity-purified anti-HMW antibody preparations was opsonophagocytic
for a representative nontypeable
H. influenzae
strain that did
not express HMW1- or HMW2-like proteins. These data demonstrate that
human antibodies specific for the HMW1/HMW2-like adhesion proteins of
nontypeable
H. influenzae
are opsonophagocytic and that such
antibodies recognize epitopes shared by the HMW proteins of unrelated
nontypeable
H. influenzae
strains. These results argue for
continued investigation of the HMW1/HMW2-like proteins as potential
vaccine candidates for prevention of disease due to
nontypeable
H.
influenzae
.
In Situ Studyof Abundant Expression of Proinflammatory Chemokines and Cytokines inPulmonary Granulomas That Develop in Cynomolgus MacaquesExperimentally Infected withMycobacteriumtuberculosis
Authors: Fuller Craig L. , Flynn JoAnne L. , Reinhart Todd A.
Published: November 2003

Tuberculosis remains a major public health problem worldwide. Chemokines and cytokines organize and direct infiltrating cells to sites of infection, and these molecules likely play crucial roles in granuloma formation and maintenance. To address this issue, we used in situ hybridization (ISH) to measure chemokine and cytokine mRNA expression levels and patterns directly in lung tissues from cynomolgus macaques (Macaca fascicularis) experimentally infected with a low dose of virulentMycobacterium tuberculosis. We examined more than 300 granulomas and observed abundant expression of gamma interferon (IFN-γ)-inducible chemokine mRNAs (CXCL9/monokine induced by IFN-γ, CXCL10/IFN-γ-inducible protein, and CXCL11/IFN-γ-inducible T-cell α-chemoattractant) within solid and caseous granulomas, and there was only minimal expression in nongranulomatous regions of tissue. The mRNA expression patterns of IFN-γ and tumor necrosis factor alpha were examined in parallel, and the results revealed that cytokine mRNA+cells were abundant and generally localized to the granulomas. Mycobacterial 16S rRNA expression was also measured by ISH, and the results revealed that there was localization predominantly to the granulomas and that the highest signal intensity was in caseous granulomas. We observed several granulomatous lesions with exceptionally high levels of RNA for mycobacterial 16S rRNA, IFN-γ, and IFN-γ-inducible chemokines, suggesting that the local presence of mycobacteria is partially responsible for the upregulation of IFN-γ-inducible chemokines and recruitment of CXCR3+cells, which were also abundant in granulomatous lesions. These results suggest that expression of CXCR3 ligands and the subsequent recruitment of CXCR3+cells are involved in granuloma formation and maintenance.
Attenuated Signature-Tagged Mutagenesis Mutants of
Brucella melitensis
Identified during the Acute Phase ofInfection inMice
Authors: Lestrate P. , Dricot A. , Delrue R.-M. , Lambert C. , Martinelli V. , De Bolle X. , Letesson J.-J. , Tibor A.
Published: November 2003

For
this study, we screened 1,152 signature-tagged mutagenesis mutants of
Brucella melitensis
16M in a mouse model of infection and
found 36 of them to be attenuated in vivo. Molecular characterization
of transposon insertion sites showed that for four mutants, the
affected genes were only present in
Rhizobiaceae
. Another
mutant contained a disruption in a gene homologous to
mosA
,
which is involved in rhizopine biosynthesis in some strains of
Rhizobium
, suggesting that this sugar may be involved in
Brucella
pathogenicity. A mutant was disrupted in a gene
homologous to
fliF
, a gene potentially coding for the MS ring,
a basal component of the flagellar system. Surprisingly, a mutant was
affected in the
rpoA
gene, coding for the essentialα
-subunit of the RNA polymerase. This disruption leaves a
partially functional protein, impaired for the activation of
virB
transcription, as demonstrated by the absence of
induction of the
virB
promoter in the
Tn
5
::
rpoA
background. The results
presented here highlight the fact that the ability of
Brucella
to induce pathogenesis shares similarities with the molecular
mechanisms used by both
Rhizobium
and
Agrobacterium
to colonize their
hosts.
Prophage Induction and Expression of Prophage-EncodedVirulence Factors in Group A
Streptococcus
Serotype M3 StrainMGAS315
Authors: Banks David J. , Lei Benfang , Musser James M.
Published: November 2003

The
genome of the highly virulent group A
Streptococcus
(GAS)
serotype M3 strain MGAS315 has six prophages that encode six proven or
putative virulence factors. We examined prophage induction and
expression of prophage-encoded virulence factors by this strain under
in vitro conditions inferred to approximate in vivo conditions.
Coculture of strain MGAS315 with Detroit 562 (D562) human epithelial
pharyngeal cells induced the prophage encoding streptococcal pyrogenic
exotoxin K (SpeK) and extracellular phospholipase
A
2
(Sla) and the prophage encoding streptodornase
(Sdn). Increased gene copy numbers after induction correlated with
increased
speK
,
sla
, and
sdn
transcript
levels. Although
speK
and
sla
are located
contiguously in prophage Φ315.4, these genes were transcribed
independently. Whereas production of immunoreactive SpeK was either
absent or minimal during coculture of GAS with D562 cells, production
of immunoreactive Sla increased substantially. In contrast, despite a
lack of induction of the prophage encoding speA during
coculture of GAS with D562 cells, the
speA
transcript level
and production of immunoreactive streptococcal pyrogenic exotoxin A
(SpeA) increased. Exposure of strain MGAS315 to hydrogen peroxide, an
oxidative stressor, induced the prophage encoding mitogenic factor 4
(MF4), and there was a concomitant increase in the
mf4
transcript. All prophages of strain MGAS315 that encode virulence
factors were induced during culture with mitomycin C, a DNA-damaging
agent. However, the virulence factor gene transcript levels and
production of the encoded proteins decreased after mitomycin C
treatment. Taken together, the results indicate that a complex
relationship exists among environmental culture conditions, prophage
induction, and production of prophage-encoded virulence
factors.
Infection and Inflammation in Skeletal Muscle fromNonhuman Primates Infected with Different Genospecies of theLyme Disease SpirocheteBorreliaburgdorferi
Authors: Cadavid Diego , Bai Yunhong , Dail Donna , Hurd Marie , Narayan Kavi , Hodzic Emir , Barthold Stephen W. , Pachner Andrew R.
Published: November 2003

Lyme borreliosis is a multisystemic disease caused by various genospecies of the spirocheteBorrelia burgdorferi. To investigate muscle involvement in the nonhuman primate (NHP) model of Lyme disease, 16 adultMacaca mulattaanimals inoculated with strain N40 ofB. burgdorferisensu strictu by syringe or by tick bite or with strain Pbi ofB. burgdorferigenospeciesgariniiby syringe were studied. Animals were necropsied while immunosuppressed on day 50 (two animals each inoculated withB.burgdorferiN40 by syringe and withB.gariniiPbi by syringe) or on day 90, 40 days after immunosuppression had been discontinued (four animals each inoculated with strain N40 by syringe, with strain N40 by tick bite, and with strain Pbi by syringe). Skeletal muscles removed at necropsy were studied by (i) microscopic examination of hematoxylin-eosin-stained sections for inflammation and tissue injury; (ii) immunohistochemical and digital image analyses for antibody and complement deposition and cellular inflammation; (iii) Western blot densitometry for the presence of antibodies; and (iv) reverse transcription-PCR for measurement of the spirochetal load or C1q (the first component of the complement cascade) synthesis. The results showed that N40 was more infectious for NHPs than Pbi. NHPs inoculated with N40 but not with Pbi developed myositis. The inflammation in skeletal muscle was more severe in NHPs inoculated with N40 by syringe than in those inoculated by tick bite. The predominant cells in the inflammatory infiltrate were T cells and plasma cells. The deposition of antibody and complement in inflamed muscles from N40-inoculated NHPs was significantly higher than that in Pbi-inoculated NHPs. The spirochetal load was very high in the two N40-inoculated NHPs examined while they were immunosuppressed but decreased to minimal levels in the NHPs when immunocompetence was restored. We conclude that myositis can be a prominent feature of Lyme borreliosis depending on the infecting organism and host immune status.