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The coronavirus disease 2019 (COVID-19) pandemic caused challenges in the management of patients living with multiple sclerosis (PLwMS). We investigated the occurrence and severity of COVID-19 infection post-vaccination among PLwMS treated with ocrelizumab and enrolled in the Maccabi Health Services (MHS) (n = 289) or followed at the Hadassah Medical Center (HMC) (n = 80) in Israel. Most patients were fully vaccinated (MHS n = 218; HMC n = 76) and confirmed infection post-vaccination was low (3.7% and 2.6%, respectively). MHS: infection was more severe (hospitalization/intensive care unit/death) in non-vaccinated (33.3%) vs vaccinated patients (25%). HMC: one vaccinated patient required hospitalization with COVID-19 vs two unvaccinated patients. These data from two Israel cohorts suggest that occurrence of COVID-19 after mRNA vaccination is low and limited in severity.

Tocilizumab is an interleukin 6 (IL-6) receptor antagonist used treat moderate to severe active rheumatoid arthritis (RA). Both intravenous (IV) and subcutaneous (SC) routes are approved for the treatment of adults with RA.To evaluate SC tocilizumab in a real-life clinical setting.Our study was a multi-center, open-label, single-arm study. Participants were adults with a diagnosis of active RA, previously treated with disease-modifying antirheumatic drugs (DMARDs), with or without biologic agents. Participants received a weekly SC injection of tocilizumab 162 mg as monotherapy or in combination with methotrexate or DMARDs for 24 weeks. Efficacy, safety, and immunogenicity were assessed.Treatment of 100 patients over 24 weeks resulted in improvement in all efficacy parameters assessed: Clinical Disease Activity Index, Disease Activity Score using 28 joint counts and erythrocyte sedimentation rate, American College of Rheumatology response scores, Simplified Disease Activity Index, tender and swollen joint counts, and patient-reported outcomes including fatigue, global assessment of disease activity, pain, and Health Assessment Quality of Life Disease Index. Improvement was achieved as early as the second week of treatment. There were 473 adverse events (AEs)/100 patient-years (PY) and 16.66 serious AEs/100 PY. The most common AEs were neutropenia (12%), leukopenia (11%), and increased hepatic enzymes (11%). Of a total of 42 PY, the rates of serious infections and AEs leading to discontinuation were 4.8, and 11.9 events/100 PY, respectively.The safety, tolerability, and efficacy profile of tocilizumab SC were comparable to those reported in other studies evaluating the IV and SC routes of administration.

The high-temperature requirement chaperone/protease (HtrA) is involved in the stress response of the anthrax-causing pathogen Bacillus anthracis. Resilience to oxidative stress is essential for the manifestation of B. anthracis pathogenicity. Here, we announce transcriptome data sets detailing global gene expression in B. anthracis wild-type and htrA-disrupted strains following H2O2-induced oxidative stress.

The high-temperature requirement chaperone/protease (HtrA) is involved in the stress response of the anthrax-causing pathogen Bacillus anthracis . Resilience to oxidative stress is essential for the manifestation of B. anthracis pathogenicity. Here, we announce transcriptome data sets detailing global gene expression in B. anthracis wild-type and htrA -disrupted strains following H 2 O 2 -induced oxidative stress.

Background Mood disorders, such as anxiety and depression, are extremely prevalent among patients with rheumatoid arthritis (RA). In this study, we assessed the impact of treatment with tocilizumab (TCZ), an IL-6 antagonist, upon anxiety and depressive symptoms in a cohort of RA patients. Materials and methods Study participants were adults diagnosed with RA who received a weekly subcutaneous injection of tocilizumab for 24 weeks. We used the Hamilton Depression (HDRS) and Anxiety (HAMA) scores in order to assess the severity of depression and anxiety, respectively. RA disease activity indices and depression and anxiety levels were assessed at baseline, 4 weeks and study completion. Results Ultimately, 91 patients were included in the study. The mean age was 54 years, and the majority were female (79%). The mean score in all disease activity indices as well as depression and anxiety levels decreased dramatically from baseline to study completion. Sixty patients (66%) demonstrated a significant decrease in anxiety and/or depression levels. When logistic regression was performed, an HDRS score indicative of depression at study baseline demonstrated an independent association with a significant psychiatric response whilst older age and increased baseline weight were negatively associated. HAMA and HDRA scores correlated with the following RA disease activity parameters, respectively; HAQ-DI (r = .4, .42), DAS28 (r = .29, .32) and CDAI (0.28 and 0.33), all of them were statistically significant (P Conclusions This study has demonstrated a favourable impact of TCZ therapy on parameters reflecting depression and anxiety severity in patients with RA.

Ricin, a highly lethal plant-derived toxin, is a potential biological threat agent due to its high availability, ease of production and the lack of approved medical countermeasures for post-exposure treatment. To date, no specific ricin receptors were identified. Here we show for the first time, that the low density lipoprotein receptor-related protein-1 (LRP1) is a major target molecule for binding of ricin. Pretreating HEK293 acetylcholinesterase-producer cells with either anti-LRP1 antibodies or with Receptor-Associated Protein (a natural LRP1 antagonist), or using siRNA to knock-down LRP1 expression resulted in a marked reduction in their sensitivity towards ricin. Binding assays further demonstrated that ricin bound exclusively to the cluster II binding domain of LRP1, via the ricin B subunit. Ricin binding to the cluster II binding domain of LRP1 was significantly reduced by an anti-ricin monoclonal antibody, which confers high-level protection to ricin pulmonary-exposed mice. Finally, we tested the contribution of LRP1 receptor to ricin intoxication of lung cells derived from mice. Treating these cells with anti-LRP1 antibody prior to ricin exposure, prevented their intoxication. Taken together, our findings clearly demonstrate that the LRP1 receptor plays an important role in ricin-induced pulmonary intoxications.

Abrin and ricin are potent AB toxins, which are considered biological threats. To date, there are no approved treatments against abrin or ricin intoxications. Previously, we showed that the administration of polyclonal anti-abrin antibodies to mice that were intranasally exposed to abrin, even very late post-exposure, conferred an exceedingly high-level of protection, while following ricin intoxication, similar treatment with anti-ricin antibodies resulted in negligible survival rates. To probe this unexpected difference in protection ability, we first examined whether the efficient anti-abrin-induced protection was due to neutralization of the A-subunit responsible for the catalytic effect, or of the B-subunit, which enables binding/internalization, by evaluating the protection conferred by antibodies directed against one of the two subunits. To this end, we generated and immunized rabbits with chimeric toxins containing a single abrin subunit, AabrinBricin in which abrin A-subunit was linked to ricin B-subunit, and AricinBabrin in which ricin A-subunit is linked to abrin B-subunit. Here, we show that antibodies raised against either AabrinBricin or AricinBabrin conferred exceptionally high protection levels to mice following intranasal exposure to a a lethal dose of abrin, suggesting that the high level of protection conferred by anti-abrin antibodies is not related to the neutralization of a particular subunit.

Ricin, a highly potent plant-derived toxin, operates by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. Recently, we presented the TRISOL [Truncated RNA Identification by Synthetic Oligonucleotide Ligation] method for the highly sensitive detection of exposures to type II ribosome-inactivating proteins such as ricin in clinical samples, based on the specific amplification of truncated cDNA molecules formed on ricin-dependent depurinated 28S rRNA templates. In the present study, we demonstrate the application of the TRISOL method to detect ricin exposure in blood samples. Sera collected from ricin-intoxicated animals was tested for the presence of either active ricin or ricin-induced depurinated 28S rRNA. Active ricin in serum samples from mice intranasally or intraperitoneally exposed to ricin was detected by the TRISOL method following incubation with a ribosomal-rich reticulocyte lysate preparation. For the detection of depurinated 28S rRNA in the serum, cell-free RNAs were isolated from the sera of mice and pigs at different time points following ricin intoxication by different exposure routes and analyzed for the presence of depurinated 28S rRNA. The TRISOL method allowed sensitive detection of both active ricin and host ribosomal damage in blood samples. Active ricin was detected at both early and late time points after ricin intoxication by all routes of exposure tested. Depurinated 28S rRNA, detected at later time points after systemic and oral intoxications, enables the determination of ricin-induced damage to cells of the exposed subject.

Type II ribosome inactivating proteins (RIPs II) such as ricin, exert their cytotoxic effect by depurinating a specific adenosine within the 28S rRNA, which in turn leads to inhibition of protein synthesis and cell death. Therapeutic intervention in case of exposure to RIP II toxins, requires the development of a specific and sensitive method for the detection of the active toxin in biological samples. Here, we describe the development of a highly sensitive assay for the detection of ricin, based on the biological activity of the toxin. We exploited the fact that, when ricin-induced depurinated 28S rRNA serves as a template in a reverse transcription reaction, cDNA elongation is prematurely terminated at the depurinated site, leading to the formation of truncated cDNA molecules. To allow specific amplification of the truncated cDNA, an unrelated synthetic single strand DNA molecule was appended to its 3′ end. This chimeric ligation product was then amplified in a quantitative real-time polymerase chain-reaction, utilizing a pair of primers, one complementing its truncated cDNA sequence, and the other complementing its synthetic single strand DNA sequence. The unique method described here detected ricin at concentrations as low as 5 pg/mL within 5 h, allowed toxin identification in biological samples from pulmonary-intoxicated mice and pigs even when collected at late time-points (30–72 h) and was also found to be highly effective in detecting ricin after intraperitoneal exposure. The method developed in this study is well-suited for detecting catalytically-active ricin in biological samples.

Pulmonary exposure to the plant toxin ricin leads to respiratory insufficiency and death. To date, in-depth study of acute respiratory distress syndrome (ARDS) following pulmonary exposure to toxins is hampered by the lack of an appropriate animal model. To this end, we established the pig as a large animal model for the comprehensive study of the multifarious clinical manifestations of pulmonary ricinosis. Here, we report for the first time, the monitoring of barometric whole body plethysmography for pulmonary function tests in non-anesthetized ricin-treated pigs. Up to 30 h post-exposure, as a result of progressing hypoxemia and to prevent carbon dioxide retention, animals exhibited a compensatory response of elevation in minute volume, attributed mainly to a large elevation in respiratory rate with minimal response in tidal volume. This response was followed by decompensation, manifested by a decrease in minute volume and severe hypoxemia, refractory to oxygen treatment. Radiological evaluation revealed evidence of early diffuse bilateral pulmonary infiltrates while hemodynamic parameters remained unchanged, excluding cardiac failure as an explanation for respiratory insufficiency. Ricin-intoxicated pigs suffered from increased lung permeability accompanied by cytokine storming. Histological studies revealed lung tissue insults that accumulated over time and led to diffuse alveolar damage. Charting the decline in PaO2/FiO2 ratio in a mechanically ventilated pig confirmed that ricin-induced respiratory damage complies with the accepted diagnostic criteria for ARDS. The establishment of this animal model of pulmonary ricinosis should help in the pursuit of efficient medical countermeasures specifically tailored to deal with the respiratory deficiencies stemming from ricin-induced ARDS., Summary: Lung injury induced by intratracheal instillation of ricin to swine exhibits the full set of parameters defining ARDS.

The plant toxin ricin is considered a biological threat agent of concern and is most toxic when inhaled. Pulmonary exposure to a lethal dose of ricin can be redressed by treatment with antiricin antibodies; however, late antitoxin intervention is of limited efficacy. This limitation is associated with overt lung damage, clinically manifested as severe pulmonary inflammation, which develops over time. Increased evidence indicates that ciprofloxacin, a broad-spectrum antimicrobial agent, possesses immunomodulatory properties. Here we demonstrate that while antiricin antibody administration at late hours after intranasal exposure to ricin confers limited protection to mice, highly efficient protection can be achieved by adding ciprofloxacin to the antibody treatment. We further demonstrate that parameters associated with lung injury, in particular, pulmonary proinflammatory cytokine production, neutrophil migration, and edema, are sharply reduced in ricin-intoxicated mice that were treated with ciprofloxacin. The presented data highlight the potential clinical application of ciprofloxacin as a beneficial immunomodulatory agent in the course of ricin intoxication.

Irrespective of its diverse etiologies, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) leads to increased permeability of the alveolar-capillary barrier, which in turn promotes edema formation and respiratory failure. We investigated the mechanism of ALI/ARDS lung hyperpermeability triggered by pulmonary exposure of mice to the highly toxic plant-derived toxin ricin. One prominent hallmark of ricin-mediated pulmonary intoxication is the rapid and massive influx of neutrophils to the lungs, where they contribute to the developing inflammation yet may also cause tissue damage, thereby promoting ricin-mediated morbidity. Here we show that pulmonary exposure of mice to ricin results in the rapid diminution of the junction proteins VE-cadherin, claudin 5, and connexin 43, belonging, respectively, to the adherens, tight, and gap junction protein families. Depletion of neutrophils in ricin-intoxicated mice attenuated the damage caused to these junction proteins, alleviated pulmonary edema, and significantly postponed the time to death of the intoxicated mice. Inhibition of matrix metalloproteinase (MMP) activity recapitulated the response to neutrophil depletion observed in ricin-intoxicated mice and was associated with decreased insult to the junction proteins and alveolar-capillary barrier. However, neutrophil-mediated MMP activity was not the sole mechanism responsible for pulmonary hyperpermeability, as exemplified by the ricin-mediated disruption of claudin 18, via a neutrophil-independent mechanism involving tyrosine phosphorylation. This in-depth study of the early stage mechanisms governing pulmonary tissue integrity during ALI/ARDS is expected to facilitate the tailoring of novel therapeutic approaches for the treatment of these diseases.

We previously demonstrated that the HtrA (High Temperature Requirement A) protease/chaperone active in the quality control of protein synthesis, represents an important virulence determinant of Bacillus anthracis. Virulence attenuation of htrA-disrupted Bacillus anthracis strains was attributed to susceptibility of &Delta, htrA strains to stress insults, as evidenced by affected growth under various stress conditions. Here, we report a comparative RNA-seq transcriptomic study generating a database of differentially expressed genes in the B. anthracishtrA-disrupted and wild type parental strains under oxidative stress. The study demonstrates that, apart from protease and chaperone activities, HtrA exerts a regulatory role influencing expression of more than 1000 genes under stress. Functional analysis of groups or individual genes exhibiting strain-specific modulation, evidenced (i) massive downregulation in the &Delta, htrA and upregulation in the WT strains of various transcriptional regulators, (ii) downregulation of translation processes in the WT strain, and (iii) downregulation of metal ion binding functions and upregulation of sporulation-associated functions in the &Delta, htrA strain. These modulated functions are extensively discussed. Fifteen genes uniquely upregulated in the wild type strain were further interrogated for their modulation in response to other stress regimens. Overexpression of one of these genes, encoding for MazG (a nucleoside triphosphate pyrophosphohydrolase involved in various stress responses in other bacteria), in the &Delta, htrA strain resulted in partial alleviation of the H2O2-sensitive phenotype.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals.

Ricin, a highly lethal toxin derived from the seeds of Ricinus communis (castor beans) is considered a potential biological threat agent due to its high availability, ease of production, and to the lack of any approved medical countermeasure against ricin exposures. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this work was to generate anti-ricin antitoxin that confers high level post-exposure protection against ricin challenge. Due to safety issues regarding the usage of ricin holotoxin as an antigen, we generated an inactivated toxin that would reduce health risks for both the immunizer and the immunized animal. To this end, a monomerized ricin antigen was constructed by reducing highly purified ricin to its monomeric constituents. Preliminary immunizing experiments in rabbits indicated that this monomerized antigen is as effective as the native toxin in terms of neutralizing antibody elicitation and protection of mice against lethal ricin challenges. Characterization of the monomerized antigen demonstrated that the irreversibly detached A and B subunits retain catalytic and lectin activity, respectively, implying that the monomerization process did not significantly affect their overall structure. Toxicity studies revealed that the monomerized ricin displayed a 250-fold decreased activity in a cell culture-based functionality test, while clinical signs were undetectable in mice injected with this antigen. Immunization of a horse with the monomerized toxin was highly effective in elicitation of high titers of neutralizing antibodies. Due to the increased potential of IgG-derived adverse events, anti-ricin F(ab&rsquo, )2 antitoxin was produced. The F(ab&rsquo, )2-based antitoxin conferred high protection to intranasally ricin-intoxicated mice, ~60% and ~34% survival, when administered 24 and 48 h post exposure to a lethal dose, respectively. In line with the enhanced protection, anti-inflammatory and anti-edematous effects were measured in the antitoxin treated mice, in comparison to mice that were intoxicated but not treated. Accordingly, this anti-ricin preparation is an excellent candidate for post exposure treatment of ricin intoxications.

Chronic fatigue is common among patients with rheumatoid arthritis (RA), affecting quality of life. Osteoporosis is a prevalent co-morbidity in RA patients.To assess the effect of long-term treatment with tocilizumab on fatigue and bone mineral density (BMD) in RA patients with inadequate response to synthetic or biologic disease-modifying anti-rheumatic drugs.In this multicenter, open-label, non-controlled, single-arm study, patients ≥ 18 years of age received intravenous tocilizumab 8 mg/kg every 4 weeks for 96 weeks. The primary outcome was the change in Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score from baseline to weeks 24, 48, 72, and 96. BMD was assessed before and 96 weeks after treatment.The study comprised 145 patients (mean age 53.4 ± 13.4 years, 83.4% women). Of these, 88 (60.7%) completed the 2 year treatment period. The mean FACIT-Fatigue score improved consistently starting from week 4 and showed a statistically significant increase of 5.0 ± 9.7, 6.8 ± 10.5, 7.3 ± 10.9, and 7.3 ± 10.4 from baseline to weeks 24, 48, 72, and 96, respectively (P0.0001). Mean BMD of femoral neck and total spine remained stable. Disease activity, acute phase reactants, and composite efficacy measures decreased during the study, while hemoglobin levels increased. Adverse events and serious adverse events were as expected for the known and previously described data.Tocilizumab therapy for 2 years significantly and clinically decreased fatigue. BMD remained stable and no new safety issue was reported.

Acetylcholinesterase (AChE) is the physiological target of organophosphate nerve agent compounds. Currently, the development of a formulation for prophylactic administration of cholinesterases as bioscavengers in established risk situations of exposure to nerve agents is the incentive for many efforts. While cholinesterase bioscavengers were found to be highly effective in conferring protection against nerve agent exposure in animal models, their therapeutic use is complicated by short circulatory residence time. To create a bioscavenger with prolonged plasma half-life, compatible with biotechnological production and purification, a chimeric recombinant molecule of HuAChE coupled to the Fc region of human IgG1 was designed. The novel fusion protein, expressed in cultured cells under optimized conditions, maintains its full enzymatic activity, at levels similar to those of the recombinant AChE enzyme. Thus, this novel fusion product retained its binding affinity toward BW284c5 and propidium, and its bioscavenging reactivity toward the organophosphate-AChE inhibitors sarin and VX. Furthermore, when administered to mice, AChE-Fc exhibits exceptional circulatory residence longevity (MRT of 6000 min), superior to any other known cholinesterase-based recombinant bioscavengers. Owing to its optimized pharmacokinetic performance, high reactivity toward nerve agents, and ease of production, AChE-Fc emerges as a promising next-generation organophosphate bioscavenger.

Anthrax is a lethal disease caused by the gram-positive spore-producing bacterium Bacillus anthracis. Live attenuated vaccines, such as the nonencapsulated Sterne strain, do not meet the safety standards mandated for human use in the Western world and are approved for veterinary purposes only. Here we demonstrate that disrupting the htrA gene, encoding the chaperone/protease HtrA (High Temperature Requirement A), in the virulent Bacillus anthracis Vollum strain results in significant virulence attenuation in guinea pigs, rabbits and mice, underlying the universality of the attenuated phenotype associated with htrA knockout. Accordingly, htrA disruption was implemented for the development of a Sterne-derived safe live vaccine compatible with human use. The novel B. anthracis SterneΔhtrA strain secretes functional anthrax toxins but is 10–104-fold less virulent than the Sterne vaccine strain depending on animal model (mice, guinea pigs, or rabbits). In spite of this attenuation, double or even single immunization with SterneΔhtrA spores elicits immune responses which target toxaemia and bacteremia resulting in protection from subcutaneous or respiratory lethal challenge with a virulent strain in guinea pigs and rabbits. The efficacy of the immune-protective response in guinea pigs was maintained for at least 50 weeks after a single immunization.

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.

Acute respiratory distress syndrome (ARDS), charactarized by an intense pulmonary inflammation involving neutrophil recruitment, is accompanied by a severe disruption of the alveolar-capillary barrier leading to life-threatening pulmonary edema. Ricin, a plant-toxin derived from the seeds of Ricinus communis, irreversibly inactivates ribosomes by site-specific depurination, thereby arresting cell protein synthesis, while its clinical manifestation following pulmonary exposure is that of rapidly developing severe ARDS. We evaluated the role of the members of tight, adherens and gap junction proteins in pulmonary edema formation in a clinically relevant mouse model of ARDS induced by ricin intoxication, since the respiratory endothelium and epithelium barrier function depends on the integrity of intercellular junctions. We found a significant reduction in Claudin5, Claudin18, VE-Cadherin and Connexin43 in mice lungs 3 hours after ricin intoxication. Neutrophil depletion markedly attenuated pulmonary edema and in combination with a matrix metalloproteinase (MMP) inhibitor restored the expression of VE-Cadherin and Connexin43, providing an evidence for MMP-dependent proteolysis of these proteins. Unlike the aforementioned proteins, disruption of Claudin18 following ricin intoxication was found to be neutrophil-independent and involved tyrosine phosphorylation. These results provide an in vivo mechanism for early impairment of the alveolar-capillary barrier by intercellular junction protein disruption, which at later time points, is enhanced by the specific loss of type II alveolar epithelial cells that no longer provide resistance to edema formation and altogether lead to respiratory failure and death.

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.

MglA (Macrophage Growth Locus A) is a pleiotropic transcription factor controlling the expression pattern of more than 100 genes in Francisella novicida, including all the ORFs located on the pathogenicity island. To further probe the role of MglA in the pathogenicity of the related strain Francisella tularensis LVS, we generated a mutant LVS strain in which the mglA locus was disrupted by insertion of a non-polar selectable marker cassette.

Significant association was found between the presence of mitral annular calcium (MAC) and aortic atheroma detected by transesophageal echocardiography. MAC may be an important marker for aortic atherosclerosis.

Natural resistance-associated macrophage protein 1 (Nramp1) is a proton/divalent cation antiporter exclusively expressed in monocyte/macrophage cells with a unique role in innate resistance to intraphagosomal pathogens. In humans, it is linked to several infectious diseases, including leprosy, pulmonary tuberculosis, visceral leishmaniasis, meningococcal meningitis, and human immunodeficiency virus as well as to autoimmune diseases such as rheumatoid arthritis and Crohn's disease. Here we demonstrate that the restricted expression of Nramp1 is mediated by the macrophage-specific transcription factor IRF-8. This factor exerts its activity via protein-protein interaction, which facilitates its binding to target DNA. Using yeast two-hybrid screen we identified Myc Interacting Zinc finger protein 1 (Miz-1) as new interacting partner. This interaction is restricted to immune cells and takes place on the promoter Nramp1 in association with PU.1, a transcription factor essential for myelopoiesis. Consistent with these data, IRF-8 knockout mice are sensitive to a repertoire of intracellular pathogens. Accordingly, IRF-8–/– mice express low levels of Nramp1 that can not be induced any further. Thus, our results explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens and point to its possible involvement in autoimmune diseases.

OBJECTIVE Leptin may be involved in the acute stress response, regulating inflammatory parameters of major importance after cardiopulmonary bypass (CPB) surgery. Critically ill patients demonstrated significant increases in leptin levels in response to stress-related cytokines (tumor necrosis factor, interleukin [IL]-1) and abolishment of the circadian rhythm of leptin secretion. We characterized the pattern of leptin secretion in the acute postoperative period in children undergoing cardiac surgery and compared the changes in leptin levels with concomitantly occurring changes in cortisol levels, IL-8, and clinical parameters. DESIGN Investigative study. SETTING University-affiliated tertiary care hospital. PARTICIPANTS AND INTERVENTIONS Twenty-nine consecutive patients, aged 6 days to 15 yrs, operated upon for the correction of congenital heart defects were studied. Surgery in 20 patients (group 1) involved conventional CPB techniques, and 9 (group 2) underwent closed-heart surgery. The time courses of leptin, cortisol, and IL-8 levels were determined. Serial blood samples were collected preoperatively, on termination of CPB, and at six time points postoperatively. Plasma was recovered immediately, aliquoted, and frozen at -70 degrees C until use. MEASUREMENTS AND MAIN RESULTS The leptin levels in group 1 decreased during CPB to 51% of baseline (p

Recently we described an unbiased bacterial whole-genome immunoinformatic analysis aimed at selection of potential CTL epitopes located in "hotspots" of predicted MHC-I binders. Applying this approach to the proteome of the facultative intra-cellular pathogen Francisella tularensis resulted in identification of 170 novel CTL epitopes, several of which were shown to elicit highly robust T cell responses. Here we demonstrate that by DNA immunization using a short DNA fragment expressing six of the most prominent identified CTL epitopes a potent and specific CD8+ T cell responses is being induced, to all encoded epitopes, a response not observed in control mice immunized with the DNA vector alone Moreover, this CTL-specific mediated immune response prevented disease development, allowed for a rapid clearance of the bacterial infection and provided complete protection against lethal challenge (10LD50) with F. tularensis holarctica Live Vaccine Strain (LVS) (a total to 30 of 30 immunized mice survived the challenge while all control DNA vector immunized mice succumbed). Furthermore, and in accordance with these results, CD8 deficient mice could not be protected from lethal challenge after immunization with the CTL-polyepitope. Vaccination with the DNA poly-epitope construct could even protect mice (8/10) against the more demanding pulmonary lethal challenge of LVS. Our approach provides a proof-of-principle for selecting and generating a multi-epitpoe CD8 T cell-stimulating vaccine against a model intracellular bacterium.

Twenty subjects identified filtered pictures of previously learned target stimuli. Five filters were utilized: 3 two-octave wide band-pass and 2 complementary (same cutoff) high- and low-pass. Response times and per cent errors were used to assess performance. The filtered pictures were presented at two sizes: to ten subjects at twice the size presented to the other ten. The results indicated that higher spatial frequencies contribute more to the identification task than do the low spatial frequencies, but also that neither low nor very high frequencies are redundant for identification. It was also seen that both the proximal (c/deg) and the distal (c/picture) scales of spatial frequency measurement are involved in the identification process.

Twelve subjects performed a complex task, detecting and recognizing small targets presented at infinity, while simultaneously monitoring a virtual image display (VID). The VID was presented at one of four optical distances (2.0, 0.5, 0.0, and-0.5 D). Optical distance was found to affect detection and recognition performance, mainly at the extreme value of 2.0 D. Interactions between optical distance and grouping of subjects according to measures of accommodation indicated that the three other optical distances affect performance differentially. The subjects' resting position of accommodation (RPA) and a combined measure of accommodation range and RPA were significantly correlated with performance (0.58). A narrowing of the functional visual field (complete misses of peripheral targets) was found at the 2.0-D optical distance for all subjects, and at the other optical distances for the subjects with an inferior accommodative mechanism (i.e., a near RPA and/or a narrow accommodation range).