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Abstract Background Dysregulation of the immune system has been shown to occur during spaceflight, although the detailed nature of the phenomenon and the clinical risks for exploration class missions have yet to be established. Also, the growing clinical significance of immune system evaluation combined with epidemic infectious disease rates in third world countries provides a strong rationale for the development of field-compatible clinical immunology techniques and equipment. In July 2002 NASA performed a comprehensive immune assessment on field team members participating in the Haughton-Mars Project (HMP) on Devon Island in the high Canadian Arctic. The purpose of the study was to evaluate the effect of mission-associated stressors on the human immune system. To perform the study, the development of techniques for processing immune samples in remote field locations was required. Ten HMP-2002 participants volunteered for the study. A field protocol was developed at NASA-JSC for performing sample collection, blood staining/processing for immunophenotype analysis, whole-blood mitogenic culture for functional assessments and cell-sample preservation on-location at Devon Island. Specific assays included peripheral leukocyte distribution; constitutively activated T cells, intracellular cytokine profiles, plasma cortisol and EBV viral antibody levels. Study timepoints were 30 days prior to mission start, mid-mission and 60 days after mission completion. Results The protocol developed for immune sample processing in remote field locations functioned properly. Samples were processed on Devon Island, and stabilized for subsequent analysis at the Johnson Space Center in Houston. The data indicated that some phenotype, immune function and stress hormone changes occurred in the HMP field participants that were largely distinct from pre-mission baseline and post-mission recovery data. These immune changes appear similar to those observed in astronauts following spaceflight. Conclusion The immune system changes described during the HMP field deployment validate the use of the HMP as a ground-based spaceflight/planetary exploration analog for some aspects of human physiology. The sample processing protocol developed for this study may have applications for immune studies in remote terrestrial field locations. Elements of this protocol could possibly be adapted for future in-flight immunology studies conducted during space missions.

Abstract Background Studies examining herpesvirus-herpesvirus (cytomegalovirus (CMV)-Epstein-Barr virus (EBV)) interactions are limited, and many of the studies have been clinical observations suggesting such an interaction exists. This report aims to examine the in vitro susceptibilities of BJAB-B1 and P3HR-1 cells (EBV positive Burkitt's lymphoma B-cell lines) to a CMV superinfection; and show that EBV reactivation occurs after CMV superinfects these cell lines. Results The BJAB-B1 and P3HR-1 cells were observed to be susceptible to a CMV superinfection by the detection of the major immediate early (MIE) viral transcript and protein (p52) expression. The BZLF1 transcript was observed in both cell lines superinfected with CMV, indicating EBV reactivation. BZLF1 protein was observed in the BJAB-B1 cells. Antigen detection was not performed in the P3HR-1 cells. Conclusion The results from the in vitro superinfections support the in vivo studies suggesting a CMV infection is related to an EBV reactivation and suggests that CMV may be important as a co-factor in EBV pathogenesis in the immunocompromised patient.

We evaluated the performance of the Luminex ARIES® C. difficile Assay on 984 stool specimens prospectively collected from patients being tested for CDI at 4 clinical laboratories in the United States. Results were compared to direct and enriched toxigenic culture. Positive percent agreement (PPA) of the ARIES® C. difficile Assay was 98.1% versus direct toxigenic culture, and sensitivity versus direct plus enriched toxigenic culture was 90.5%. Negative percent agreement (NPA) of the ARIES® C. difficile Assay against direct culture was 92.6%, and specificity versus direct plus enriched toxigenic culture was 95.8%. The ARIES® C. difficile Assay was also compared to the results of routine (molecular, antigen, and/or toxin) methods for C. difficile testing used at each institution. The PPA of the ARIES® C. difficile Assay ranged from 82.9% to 100%. NPA values against these commercial assays ranged from 94.5% to 100%. [ABSTRACT FROM AUTHOR]

Candida auris is an emerging multidrug-resistant fungal pathogen that has been associated with nosocomial bloodstream and deep wound infections causing a high mortality rate mainly in intensive care unit (ICU) patients. Laboratories currently rely on phenotypic testing using commercial automated systems for identification of yeasts; however, this technique has often led to misidentification of C. auris to other closely related species. We developed and validated a TaqMan-based real-time PCR assay on the BD Max platform targeting ribosomal DNA (rDNA) region nucleotide sequences to quickly and accurately test for C. auris infection from culture and clinical specimens. The assay is highly specific, reproducible, and sensitive, allowing detection of as low as 1 C. auris CFU per reaction within 3 h., (Copyright © 2019 American Society for Microbiology.)

Background: Blood cultures, the gold standard for diagnosing bloodstream infections (BSIs), are insensitive and limited by prolonged time to results. The T2Bacteria Panel (T2 Biosystems) is a direct-from-blood, nonculture test that identifies the most common ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli)., Objective: To assess performance of the T2Bacteria Panel in diagnosing suspected BSIs in adults., Design: Prospective patient enrollment (8 December 2015 through 4 August 2017)., Setting: Eleven U.S. hospitals., Patients: 1427 patients for whom blood cultures were ordered as standard of care., Intervention: Paired blood culture and T2Bacteria testing., Measurements: Performance of T2Bacteria compared with a single set of blood cultures in diagnosing proven, probable, and possible BSIs caused by T2Bacteria-targeted organisms., Results: Blood culture and T2Bacteria results were positive for targeted bacteria in 3% (39 of 1427) and 13% (181 of 1427) of patients, respectively. Mean times from start of blood culture incubation to positivity and species identification were 38.5 (SD, 32.8) and 71.7 (SD, 39.3) hours, respectively. Mean times to species identification with T2Bacteria were 3.61 (SD, 0.2) to 7.70 (SD, 1.38) hours, depending on the number of samples tested. Per-patient sensitivity and specificity of T2Bacteria for proven BSIs were 90% (95% CI, 76% to 96%) and 90% (CI, 88% to 91%), respectively; the negative predictive value was 99.7% (1242 of 1246). The rate of negative blood cultures with a positive T2Bacteria result was 10% (146 of 1427); 60% (88 of 146) of such results were associated with probable (n = 62) or possible (n = 26) BSIs. If probable BSIs and both probable and possible BSIs were assumed to be true positives missed by blood culture, per-patient specificity of T2Bacteria was 94% and 96%, respectively., Limitation: Low prevalence of positive blood cultures, collection of a single set of culture specimens, and inability of T2Bacteria to detect nontargeted pathogens., Conclusion: The T2Bacteria Panel rapidly and accurately diagnoses BSIs caused by 5 common bacteria., Primary Funding Source: T2 Biosystems.

Respiratory viral infections are associated with a wide range of acute syndromes and infectious disease processes in children and adults worldwide. Many viruses are implicated in these infections, and these viruses are spread largely via respiratory means between humans but also occasionally from animals to humans. This article is an American Society for Microbiology (ASM)-sponsored Practical Guidance for Clinical Microbiology (PGCM) document identifying best practices for diagnosis and characterization of viruses that cause acute respiratory infections and replaces the most recent prior version of the ASM-sponsored Cumitech 21 document, Laboratory Diagnosis of Viral Respiratory Disease , published in 1986. The scope of the original document was quite broad, with an emphasis on clinical diagnosis of a wide variety of infectious agents and laboratory focus on antigen detection and viral culture. The new PGCM document is designed to be used by laboratorians in a wide variety of diagnostic and public health microbiology/virology laboratory settings worldwide. The article provides guidance to a rapidly changing field of diagnostics and outlines the epidemiology and clinical impact of acute respiratory viral infections, including preferred methods of specimen collection and current methods for diagnosis and characterization of viral pathogens causing acute respiratory tract infections. Compared to the case in 1986, molecular techniques are now the preferred diagnostic approaches for the detection of acute respiratory viruses, and they allow for automation, high-throughput workflows, and near-patient testing. These changes require quality assurance programs to prevent laboratory contamination as well as strong preanalytical screening approaches to utilize laboratory resources appropriately. Appropriate guidance from laboratorians to stakeholders will allow for appropriate specimen collection, as well as correct test ordering that will quickly identify highly transmissible emerging pathogens., (Copyright © 2018 American Society for Microbiology.)

The aim of this study was to develop a multiplex PCR test to detect Achromobacter xylosoxidans (AX), Burkholderia cepacia (BC), Pseudomonas aeruginosa (PSA) and Stenotrophomonas maltophilia (SM) directly from CF patient's respiratory samples using the open mode of the BD MAX™ System. A total of 402 CF respiratory samples were evaluated by culture and PCR. Specific sets of primers and probes for each target were designed in-house. Out of 402 samples tested, 196 were identified as negative and 206 as positive by culture for AX, PSA, BC and SM. Among culture positive samples, PCR detected 21/27 AX, 4/5 BC, 138/140 PSA and 29/34 SM. In addition, PCR assay identified 35 samples as positive that were initially negative by culture for those 4 targets. The CF BDM test proved to be an excellent tool to detect AX, BC, PSA and SM by real-time PCR on an automated platform.

Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) infections are a burden on the health care system. Clinical laboratories play a key role in reducing this burden, as the timely identification of MRSA colonization or infection facilitates infection control practices that are effective at limiting invasive MRSA infections. The Xpert MRSA NxG assay recently received FDA clearance for the direct detection of MRSA from nasal swabs. This multicenter study evaluated the clinical performance characteristics of the Xpert MRSA NxG assay with prospectively collected rayon nasal swabs ( n = 1,103) and flocked swab (ESwab) nasal specimens ( n = 846). Culture-based identification methods and antimicrobial susceptibility testing were used as the reference standards for comparison. According to the reference method, the positivity rates for MRSA in the population evaluated were 11.1% (122/1,103) for rayon swabs and 11.6% (98/846) for flocked swabs. The overall sensitivity and specificity of the rayon swabs were 91.0% (95% confidence interval [CI], 84.6 to 94.9%) and 96.9% (95% CI, 95.7 to 97.8%), respectively, across eight testing sites. The flocked swab specimens were 92.9% sensitive (95% CI, 86.0 to 96.5%) and 97.6% specific (95% CI, 96.2 to 98.5%) for MRSA detection across six testing sites. The sensitivity and specificity of the combined flocked and rayon swab data were 91.8% (95% CI, 87.4 to 94.8%) and 97.2% (95% CI, 96.3 to 97.9%), respectively. The positive predictive value (PPV) for rayon swabs was 78.7%, versus 83.5% for ESwabs. The negative predictive values (NPVs) for rayon swabs and ESwab specimens were 98.9% and 99.1%, respectively. In conclusion, the Xpert MRSA NxG assay is a sensitive and specific assay for the direct detection of MRSA from nasal swab specimens., (Copyright © 2017 American Society for Microbiology.)

A new multiplex PCR test was designed to detect Mycobacterium chelonae, Mycobacterium abscessus group, and Mycobacterium fortuitum complex on the BD MAX System. A total of 197 clinical samples previously submitted for mycobacterial culture were tested using the new protocol. Samples were first treated with proteinase K, and then each sample was inoculated into the BD MAX Sample Buffer Tube. Extraction and multiplex PCR were performed by the BD MAX System, using the BD MAX ExK TNA-3 extraction kit and BD TNA Master Mix, along with specific in-house designed primers and probes for each target. The limit of detection of each target, as well as specificity, was evaluated. Of 197 clinical samples included in this study, 133 were positive and 60 were negative for mycobacteria by culture, and another 4 negative samples were spiked with M. chelonae ATCC 35752. The new multiplex PCR on the BD MAX had 97% concordant results with culture for M. abscessus group detection, 99% for M. chelonae, and 100% for M. fortuitum complex. The new multiplex PCR test performed on the BD MAX System proved to be a sensitive and specific test to detect M. chelonae, M. abscessus group, and M. fortuitum complex by real-time PCR on an automated sample-in results-out platform., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Group B Streptococcus detection directly from Copan ESwab collected samples, using the BD Max GBS assay, was evaluated on receipt in the laboratory and after 24 h at room temperature. Results were compared to those using Lim broth enrichment PCR and culture. No significant difference was observed between 24 h ESwab and Lim broth PCRs., (Copyright © 2016 Silbert et al.)

A multiplex real-time PCR was validated on the BD Max open system to detect different Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium spp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The BD Max MRSAXT and the BD Max StaphSR assays were validated for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in ESwab samples. In addition, the BD Max StaphSR assay was evaluated for its ability to detect and differentiate S. aureus and MRSA in the same sample. A total of 255 ESwab samples collected from the anterior nares of patients were tested by each of three BD Max assays, including the BD Max MRSA first-generation assay. The results were compared to those of direct and enrichment culture. Additionally, a challenge panel comprising 14 control strains was evaluated to determine the ability of these assays to correctly identify MRSA and also appropriately differentiate S. aureus by the BD Max StaphSR assay. Out of 255 clinical samples tested, 161 were negative and 30 were positive for MRSA, and 45 were positive for S. aureus (by BD Max StaphSR) and negative for MRSA by all three PCR assays and culture. Nineteen samples had discrepant results; all of them were retested by additional laboratory testing. All strains from the challenge panel were correctly identified or excluded by the BD Max MRSAXT and BD Max StaphSR assays. The results showed that the BD Max StaphSR and the BD MRSAXT assays have excellent sensitivity (94.3%) and specificity (97.7%) for detecting MRSA. The BD Max StaphSR assay demonstrated excellent sensitivity (96.4%) and specificity (93.6%) for detecting S. aureus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A comparison between the BD MAX MRSA and Xpert MRSA assays was performed using 239 nares samples. A 97.9% overall agreement between the two molecular assays was observed. The BD MAX MRSA assay proved to be a reliable alternative for a highly automated system to detect methicillin-resistant Staphylococcus aureus (MRSA) in patient nares samples., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The ESwab system (Copan Diagnostics) was evaluated as a nasopharyngeal specimen collection device to be used for methicillin-resistant Staphylococcus aureus (MRSA) detection by the GeneXpert and BD Max MRSA assays. Different MRSA strains and dilutions of each strain were tested in triplicate. ESwabs proved to be a suitable collection system for the two assays tested., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Purpose: In myeloma, B cells and plasma cells show a clonal relationship. Clonotypic B cells may represent a tumor-initiating compartment or cancer stem cell responsible for minimal residual disease in myeloma., Experimental Design: We report a study of 58 patients with myeloma at time of diagnosis or relapse. B cells in bone marrow were evaluated by multicolor flow cytometry and sorting. Clonality was determined by light chain and/or immunoglobulin chain gene rearrangement PCR. We also determined aldehyde dehydrogenase activity and colony formation growth. Drug sensitivity was tested with conventional and novel agents., Results: Marrow CD19+ cells express a light chain identical to plasma cells and are therefore termed light chain restricted (LCR). The LCR B-cell mass is small in both newly diagnosed and relapsed patients (≤ 1%). Few marrow LCR B cells (~10%) are CD19+/CD34+, with the rest being more differentiated CD19+/CD34- B cells. Marrow LCR CD19+ B cells exhibit enhanced aldehyde dehydrogenase activity versus healthy controls. Both CD19+/CD34+ and CD19+/CD34- cells showed colony formation activity, with colony growth efficiency optimized when stroma-conditioned medium was used. B-cell progenitors showed resistance to melphalan, lenalidomide, and bortezomib. Panobinostat, a histone deacetylase inhibitor, induced apoptosis of LCR B cells and CD138+ cells. LCR B cells are CD117, survivin, and Notch positive., Conclusions: We propose that antigen-independent B-cell differentiation stages are involved in disease origination and progression in myeloma. Furthermore, investigations of myeloma putative stem cell progenitors may lead to novel treatments to eradicate the potential reservoir of minimal residual disease., (©2012 AACR.)

Marijuana cannabinoids, the endocannabinoids, and cannabinoid cell receptors have been shown to play important roles in immune regulation particularly as potent modulators of anti-inflammatory cytokines. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB(2)), which is predominantly expressed in B lymphocytes. However, the promoter region and mechanisms of CB(2) gene regulation are unknown in this immune cell type. Utilizing a combination of bioinformatics, 5' rapid amplification of cDNA ends (5' RACE), real-time reverse transcription-polymerase chain reaction, DNA sequencing, and luciferase reporter assays, we show that human B cells express one CB(2) transcript while mouse B cells express three CB(2) transcripts, with specific transcript selection occurring during B cell activation by lipopolysaccharide. Alignment of our sequenced RACE products to either the mouse or human genome, along with the GenBank submitted mRNA sequences, revealed that the transcripts we isolated contained previously unidentified transcriptional start sites (TSS). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB(2) exon 1 transcripts showed an eightfold increase of promoter activity over baseline. These data show for the first time that human B cells use only one TSS for CB(2) while mouse B cells use multiple TSSs and that the mouse TSSs are in a genomic area with promoter activity, thus suggesting the location of the gene promoter region. Defining these TSSs also provides clues to the various gene regulatory factors involved in the expression of CB(2) during B cell activation.

Objectives: To evaluate the efficacy of antibiotic-impregnated polymethylmethacrylate (PMMA) beads in eradication of an arterial prosthetic graft methicillin-resistant Staphylococcus aureus (MRSA) biofilm in an experimental animal model., Methods: Forty rats underwent subcutaneous implantation of a MRSA-colonized arterial polytetrafluoroethylene (PTFE) 1 x 1 cm wafer on the back. The effect of regional antibiosis produced by antibiotic PMMA bead placement adjacent to the infected PTFE wafer was determined using four 10-animal study groups: control (no antibiotic), PMMA bead with no antibiotic, PMMA bead with 10% vancomycin, and PMMA bead with 10% daptomycin. After 3 d, the PTFE wafers were explanted and quantitative biofilm cultures, expressed as colony-forming units (CFU) per graft wafer, performed using real-time polymerase chain reaction to assess MRSA eradication. No systemic antibiotic was administered. Bioassays of antibiotic bead bacteriocidal were performed by measuring zone of inhibition diameters on MRSA colonized agar culture plates prior to and following graft explantation., Results: All animal tolerated implantation of the MRSA-infected PTFE wafer and survived the 3 d until graft explantation. Quantitative biofilm cultures demonstrated a significant decrease (P < 0.01) in MRSA CFUs present on the PTFE wafer surfaces in the presence of both the vancomycin- and daptomycin-impregnated beads compared to controls and plain PMMA beads. Both vancomycin and daptomycin PMMA beads retained antibacterial activity after 3 d of implantation with decrease in zones of inhibition of 15% and 45%, respectively., Conclusions: Regional antibiotic delivery using an antibiotic-impregnated PMMA bead reduced the bacterial biofilm concentration in experimental subcutaneous pocket model of vascular surgical site infection. The delivery of antibiotics via a PMMA bead may be a useful adjunct in the treatment of vascular surgical site infection.

Marijuana cannabinoid treatment increases Th2 activity, and previous reports showed that B cells express the highest level of CB(2) mRNA relative to other immune cells, suggesting that cannabinoids play a critical role in B cell activation and maturation. We previously reported evidence of Th2 biasing and class switching in cannabinoid-treated and antigen-challenged mice. We now explore the possibility that cannabinoids directly influence B cell antibody class switching. Mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB(1) selective cannabinoid agonist, methanandamide, and analyzed at different days by flow cytometry for surface expression of either IgM or IgE. Cells treated with CP55940 showed an increase in expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants as analyzed by ELISA. In addition, CB(2) receptors were increased on B cells after stimulation with IL-4 and anti-CD40, and the class switching effect of CP55940 was attenuated by the CB(2) antagonist, SR144528. These results suggest that cannabinoids bias toward Th2-type immunity by directly inducing B cell class switching from IgM to IgE through a mechanism involving CB(2) receptors.

Dendritic cells (DCs) have a critical role in linking innate to adaptive immunity, and this transition is regulated by the up-regulation of costimulatory and major histocompatibility complex (MHC) molecules as well as Toll-like receptors. These changes in DCs have been observed to occur following microbial infection, and in the present study, we examined the effect of Legionella pneumophila infection on the expression of these DC markers. We showed that bone marrow-derived DC cultures from BALB/c mice infected with live L. pneumophila resulted in the up-regulation of Toll-like receptors 2 and 4 and the activation of CD40, CD86, and MHC class I/II molecules.

Background: Dysregulation of the immune system has been shown to occur during spaceflight, although the detailed nature of the phenomenon and the clinical risks for exploration class missions have yet to be established. Also, the growing clinical significance of immune system evaluation combined with epidemic infectious disease rates in third world countries provides a strong rationale for the development of field-compatible clinical immunology techniques and equipment. In July 2002 NASA performed a comprehensive immune assessment on field team members participating in the Haughton-Mars Project (HMP) on Devon Island in the high Canadian Arctic. The purpose of the study was to evaluate the effect of mission-associated stressors on the human immune system. To perform the study, the development of techniques for processing immune samples in remote field locations was required. Ten HMP-2002 participants volunteered for the study. A field protocol was developed at NASA-JSC for performing sample collection, blood staining/processing for immunophenotype analysis, whole-blood mitogenic culture for functional assessments and cell-sample preservation on-location at Devon Island. Specific assays included peripheral leukocyte distribution; constitutively activated T cells, intracellular cytokine profiles, plasma cortisol and EBV viral antibody levels. Study timepoints were 30 days prior to mission start, mid-mission and 60 days after mission completion., Results: The protocol developed for immune sample processing in remote field locations functioned properly. Samples were processed on Devon Island, and stabilized for subsequent analysis at the Johnson Space Center in Houston. The data indicated that some phenotype, immune function and stress hormone changes occurred in the HMP field participants that were largely distinct from pre-mission baseline and post-mission recovery data. These immune changes appear similar to those observed in astronauts following spaceflight., Conclusion: The immune system changes described during the HMP field deployment validate the use of the HMP as a ground-based spaceflight/planetary exploration analog for some aspects of human physiology. The sample processing protocol developed for this study may have applications for immune studies in remote terrestrial field locations. Elements of this protocol could possibly be adapted for future in-flight immunology studies conducted during space missions.

Migration and differentiation of monocytes to the intima of blood vessels may be a crucial first step in the development of atherosclerosis associated with Chlamydia (Chlamydophila) pneumoniae. However, the involvement of C. pneumoniae infection in such steps is not clear. In the present study, therefore, the differentiation-inducing activity of C. pneumoniae to monocytes was examined. Human THP-1 monocytic cell line cells were infected with C. pneumoniae, and the differentiation of monocytes to macrophages was assessed by cell morphology, phagocytic activity, and expression of a cell surface adhesion molecule. The monocytic cells infected with viable bacteria markedly differentiated into macrophages associated with diffused cell morphology, increased uptake of polystyrene beads and increased ICAM-1 (intercellular adhesion molecule 1) expression on the cell surfaces. Heat-killed bacteria did not induce any morphological changes or increase of phagocytosis, but they did induce an increase of cell surface ICAM-1 expressions in THP-1 monocytic cells. The antibiotic minocycline treatment of infected cells resulted in marked inhibition of the cell differentiation as well as C. pneumoniae growth in the cells, but not ICAM-1 expression. In addition, the experiments with human peripheral blood monocytes infected with C. pneumoniae also showed the differentiation of macrophages assessed by morphological change and phagocytic activity. These results indicate that C. pneumoniae infection may directly induce the differentiation of monocytes to macrophages. However, antigenic stimulation of monocytes with bacteria may not be sufficient for a full macrophage differentiation.

One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component. The most thoroughly studied type IV secretion system is encoded by the virB operon of Agrobacterium tumefaciens. In Bartonella henselae, 8 of the 10 virB operon genes share extensive homology and arrangement with the virB operon of A. tumefaciens. Sequencing of the region upstream of the B. henselae virB2 gene revealed a region with sequence homology to the vir box of A. tumefaciens. This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform B. henselae. Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells. Deletion of an 87-bp fragment, which contained the putative vir box from the 5' end of the promoter region, diminished intracellular induction of the reporter gene. Host cell induction of the 17-kDa antigen gene, which replaces virB5 in B. henselae, was also demonstrated at the protein level using specific antiserum. Thus, expression of the virB genes of B. henselae is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in the virB operon of A. tumefaciens.

Cannabinoid CB(2) receptor is reported to be expressed in varying amounts in different human immune subpopulations. To examine the expression pattern of CB(2) in the mouse, immune cell subpopulations were purified and studied by semiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR). CB(2) mRNA was most abundant in splenic B cells, followed by macrophages and T cells. Furthermore, CB(2) was expressed in thioglycollate-elicited peritoneal macrophages, but not in resident peritoneal macrophages. In addition to these studies on receptor expression at basal activity, CB(2) mRNA expression was also studied following immune cell activation. Bacterial lipopolysaccharide stimulation downregulated CB(2) mRNA expression in splenocyte cultures in a dose-response manner, while stimulation through cluster of differentiation 40 (CD40) using anti-CD40 antibody upregulated the response and costimulation with interleukin-4 attenuated the anti-CD40 response. These results demonstrate that CB(2) mRNA expression differs among mouse immune subpopulations similar to what is observed in human immune cells. Furthermore, the results suggest that the signaling pathways activated by lipopolysaccharide and anti-CD40 might have different effects on CB(2) mRNA expression.

Gamma interferon (IFN-gamma), a pleiotropic cytokine, is now known to be produced by macrophages as well as by NK cells, gammadelta cells, and activated T cells. The autocrine biological functions of IFN-gamma on the macrophage include the upregulation of major histocompatibility complex MHC class II and the activation to an antiviral state. In this study, the production of IFN-gamma by macrophages was demonstrated to correspond to antibacterial activity. Legionella pneumophila replicates intracellularly in thioglycolate (TG)-elicited macrophages (TG-macrophages) from A/J mice, while TG-macrophages from BALB/c mice restrict bacterial growth after an initial period of growth. BALB/c TG-macrophages were shown to express IFN-gamma mRNA at 24 and 28 h, which corresponded to the initiation of anti-L. pneumophila activity. Moreover, IFN-gammaneutralization by antibody treatment of the cultures resulted in increased L. pneumophila growth in the macrophages. In contrast, no IFN-gamma mRNA was expressed in TG-macrophages from A/J mice, where L. pneumophila grew unrestricted. As would be expected, IFN-gamma treatment decreased bacterial growth. An IFN-gamma-mediated antibacterial activity was, however, inducible in A/J macrophages by the addition of interleukin-12 following L. pneumophila infection. Thus, autocrine IFN-gamma is involved in anti-L. pneumophila activity associated with different growth patterns and appears to be important during intracellular infection.

There is significant evidence that cannabinoids have the ability to exert immunomodulatory effects. The identification of cannabinoid receptors in immune tissues has therefore led to questions about whether these immunomodulatory effects occur via these cannabinoid receptors. The cannabinoid receptor 1 (CB 1), although expressed primarily in the brain, is also expressed in lower amounts in peripheral tissues. Of interest to us is the fact that CB1 is expressed in immune tissues such as spleen, albeit at lower levels than the peripheral cannabinoid receptor, CB2. To examine the function of CBI in immune cells, activation experiments were performed using different stimuli e.g., anti-CD3, phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Io), and PMA/Io + IL-2. Whole spleen cells were cultured in the presence of different stimuli for 0, 2, 4, and 24 hours, harvested at each time point, RNA isolated, and RT-PCR performed. FACS analysis was also performed using CD69 (an early activation marker) to determine whether cells were actually being activated. Results from anti-CD3 stimulation indicated a decrease in CB1 mRNA expression following activation. CB1 mRNA expression in murine splenocytes that were stimulated with PMA/Io in the presence or absence of IL-2 was also modulated. Expression of the message was enhanced upon stimulation with PMA/Io and PMA/Io + IL-2, however, stimulation with PMA/Io + IL-2 led to a stronger increase within 2 to 4 hours with CB1 returning to at or below baseline levels by 24 hours. Expression of CD69 was detected in all stimulated samples thereby indicating that the splenocytes were becoming activated. In summary, anti-CD3 stimulation appeared to decrease CB1 mRNA expression while PMA/Io + IL-2 stimulation significantly increased CB1 mRNA expression. These results demonstrate that the expression of CB1 mRNA is modulated upon cellular activation and that this modulation is dependent on the stimulus that is used.

Cannabinoid receptor 2 (CB2) has been identified as the most abundant cannabinoid receptor subtype in the immune system. Bacterial lipopolysaccharide (LPS) is a potent stimulant of B cells, inducing proliferation and differentiation into antibody secreting cells. It has been reported that CB2 receptor expression is upregulated during human, tonsillar B cell activation through CD40. It was of interest to investigate the expression of CB2 mRNA using another B cell activator, LPS. Using northern blot analysis, we measured CB2 mRNA levels in murine splenocytes and enriched B cells. Results indicated that the 4.0 kb CB2 transcript was 2 fold higher in abundance in murine B cells than in whole splenocyte preparations. This observation confirmed data from others and from our previous RT-PCR studies that the expression of CB2 mRNA is more abundant in B cells. Upon LPS stimulation, CB2 transcripts were decreased 46% and 42% at 4 hours and 24 hours, respectively, when compared to unstimulated populations. An examination by flow cytometry of the CD69, early activation marker, on splenocytes, showed that the majority of the B cells were activated at 24 hrs. Thus, these results suggested that LPS stimulation of murine B cells caused a decrease in CB2 mRNA expression in contrast to the increase observed following human B cell stimulation through CD40.

The expression and function of cannabinoid receptor 1 (CB1) in mouse immune cells is unclear. Here we show that splenic B cells express more CB1 mRNA than T cells. Furthermore, splenocytes stimulated with the T cell mitogens, PMA/Io and anti-CD3, showed a decrease in CB1 message while cultures stimulated with the B cell mitogen, anti-CD40 antibody, showed an increase in message. In addition, co-treatment with mitogens and IL-2 uniformly caused an increase in CB1 mRNA. It is suggested that signaling pathways activated by T cell mitogens lead to decreased CB1 gene activation while pathways activated by B cell mitogens and IL-2 lead to increased CB1.

Bacterial htrA genes are typically activated as part of the periplasmic stress response and are dependent on the extracytoplasmic sigma factor rpoE. A putative promoter region, P1, of the sigma(E)-type heat-inducible promoters has previously been identified upstream of the htrA gene of Bartonella henselae. Further analysis of the htrA mRNA by primer extension demonstrated that transcription initiates from P1 and a second region downstream of P1. This second promoter region, termed P2, had no sequence identity to sigma(E)-type heat-inducible promoters. Promoter regions were cloned individually and in tandem into pANT3 upstream of a promoterless version of the green fluorescent protein (GFP) gene (gfpmut3) and transformed into B. henselae by electroporation. The contiguous promoter region containing both P1 and P2 were necessary for the optimal transcriptional activation of the htrA gene. Promoter activity at 37 degrees C was distinctively higher than at 27 degrees C. However, thermal induction at 47 degrees C did not increase expression of gfpmut3. Invasion of human microvascular endothelial cells (HMEC-1) by B. henselae resulted in the formation of well-defined vacuoles containing clusters of bacteria exhibiting marked expression of gfpmut3 transcribed from the P1-P2 region. In addition, a moderate yet significant increase in the ratio of bacterial GFP to DNA was detected for intracellular bacteria compared to extracellular bacteria, indicating upregulation of htrA upon invasion of HMEC-1. The activation of specific genes in the intracellular environment may help us better understand the novel pathogenic mechanisms used by this bacterium.

Infection of BALB/c mice with a sublethal concentration of Legionella pneumophila causes an acute disease that is resolved by innate immune responses. The infection also initiates the development of adaptive Th1 responses that protect the mice from challenge infections. To study the early responses, cytokines induced during the first 24 h after infection were examined. In the serum, interleukin-12 (IL-12) was detectable by 3 h and peaked at 10 h, while gamma interferon was discernible by 5 h and peaked at 8 h. Similar patterns were observed in ex vivo cultures of splenocytes. A transient IL-4 response was also detected by 3 h postinfection in ex vivo cultures. BALB/c IL-4-deficient mice were more susceptible to L. pneumophila infection than were wild-type mice. The infection induced higher serum levels of acute-phase cytokines (tumor necrosis factor alpha [TNF-alpha], IL-1beta, and IL-6), and reducing TNF-alpha levels with antibodies protected the mice from death. Moreover, the addition of IL-4 to L. pneumophila-infected macrophage cultures suppressed the production of these cytokines. Thus, the lack of IL-4 in the deficient mice resulted in unchecked TNF-alpha production, which appeared to cause the mortality. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that is induced by IL-4 during Listeria monocytogenes infection, was detected at between 2 and 30 h after infection. However, MCP-1 did not appear to be induced by IL-4 or to be required for the TNF-alpha regulation by IL-4. The data suggest that the early increase in IL-4 serves to regulate the mobilization of acute phase cytokines and thus controls the potential harmful effects of these cytokines.

Immunotherapy involving cytotoxic T lymphocytes (CTLs) is an attractive alternative for treatment of various malignancies, including multiple myeloma. For tumour cells to be recognized and killed by CTLs they must express cell surface major histocompatibility complex (MHC) class I molecules and the transporter associated with antigen processing (TAP). However, loss of MHC class I and the TAP protein are common among several types of solid tumours. This study assessed the expression of TAP protein (by intracellular flow cytometry) and cell surface MHC class I molecules in three human myeloma cell lines as well as the plasma cell population (CD38+ bright) in bone marrow specimens from 13 multiple myeloma patients. In all of the patients, 100% of the plasma cell population expressed both the TAP subunits and cell surface MHC class I molecules, but at varying intensities. Both TAP and MHC class I were also expressed in the three myeloma lines. Additionally, the function of the antigen transport machinery was evaluated by a peptide transporter assay in the three myeloma lines. TAP transporter activity was readily detectable in two out of three myeloma lines, whereas the diminished activity in the third cell line was completely restored by co-culturing with recombinant interferon-gamma (rIFN-gamma).

Tetrahydrocannabinol (THC) the major psychoactive component of marijuana, differentially modulates the immune response of lymphoid cells from young and old mice. This effect was previously shown when cells from mice of different ages were treated with THC in vitro. In the present study THC was given in vivo to mice of different ages. Lymphoid cells from the young and old mice had different immunologic potential in terms of ability to produce cytokines following stimulation with either Con A or anti-CD3 antibody. IL-4 and IL-10 production was consistently up-regulated in spleen cell cultures from the older animals. In addition, in vivo administration of THC up-regulated the proliferative response of lymphoid cells from young adult mice. Such enhancement was not evident for cells from older animals. Although the proliferative response of spleen cells in the old mice tended to be suppressed, statistical significance was not evident possibly because of marked variation of the responses of cells from the older mice. Since marijuana is used by persons of a wide range of ages, aging should be an important variable that must be considered when assessing the immunomodilatory effect of this drug.

A cell line was established from a mixed mullerian tumor of the ovary and designated LN1. Histopathologic analysis of the fresh tumor specimen demonstrated a highly aneuploid heterologous tumor comprised of undifferentiated mesodermal components with carcinomatous cells present as a smaller population. Long-term in vitro culture resulted in the establishment of a cell line that exhibits an epithelial-like morphology and expresses epithelial antigens cytokeratin, epithelial membrane antigen, and carcinoma antigen TAG-72. These cells also express mesenchymal intermediate filaments, vimentin, and desmin. Karyotypic analysis revealed a basic triploid pattern with multiple chromosomal abnormalities, most notably an isochromosome of the short arm of five present in three copies. Analysis of oncogene expression revealed that LN1 cells constitutively express mRNA for c-ras, c-erbB2, and p53. The expression of mRNA for cellular oncogenes correlated with the presence of corresponding oncoproteins, p21H-ras, p21K-ras, and p185erB2 and mutant p53 protein. In summary, coexpression of epithelial and mesenchymal antigens by LN1 cells lends support to the hypothesis that epithelial and mesenchymal elements comprising mixed mullerian tumors of the ovary are derived from a common stem cell precursor. Furthermore, this cell line represents a functional in vitro model to evaluate the biologic activities of these unusual and highly aggressive ovarian malignancies.

Production of T helper 1 and T helper 2 cytokines was investigated in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients by a newly described technique, detection of intracellular cytokines by flow cytometry in conjunction with immunophenotype analysis. T-cell gamma interferon (IFN-gamma) production and interleukin 10 (IL-10) production were examined after PBMC activation with T-cell mitogens at 5 and 24 h, and monocyte spontaneous production of IL-10 and production after PBMC activation with lipopolysaccharide (LPS) for 24 h were also examined. The data indicate that MS patients have decreased percentages of T cells capable of secreting IFN-gama compared with healthy controls, and this change is detectable at 5 and 24 h. the patients displaying decreased T-cell production of IFN-gamma were essentially confined to a group being treated with the newly approved drug Betaseron (berlex Labs, Cedar Knolls, N.J.), a recombinant form of IFN-beta (rIFN-beta 1b). By gating of the entire lymphocyte population, analysis of IFN-gama production in T cells (CD3+ versus that in non-T cells (CD3+) was possible. The percentage of IFN-gamma-producing lymphocytes that was made up of T cells was essentially unchanged between the Betaseron-treated patients, non-Betaseron-treated patients, and controls, indicating that the suppression of IFN-gamma production displayed by betaseron-treated MS patients was a nonspecific suppression of all IFN-gamma-producing lymphocytes as opposed to a suppression of T-cell production only. The data seem to indicate that treatment of MS with Betaseron corresponds to an inhibition of the lymphocyte's ability to produce IFN-gamma. No changes were detected in T-cell production of IL-10 at either time point. We also observed that MS patients in general appear to have small percentages of peripheral blood monocytes spontaneously producing slight but detectable levels of IL-10. No difference was seen regarding monocyte production of IL-10 after PBMC activation with LPS between MS patients and controls. Both populations responded with high percentages of monocytes producing IL-10. The data seem to indicate that treatment of MS with Betaseron, known to decrease the exacerbation rate of relapsing-remitting MS, corresponds to a suppression of peripheral blood lymphocyte production of IFN-gamma. Monocyte production of IL-10 may also play a role in regulating the disease process.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS). The disease is characterized by inflammatory lesions in the white matter of the CNS, consisting of a specific immune response to the myelin sheath. We investigated peripheral blood mononuclear cell (PBMC) cytokine production by enzyme-linked immunosorbent assays of 21 MS patients and 19 age-matched normal controls in response to the T-cell mitogen phytohemagglutinin (PHA). Peripheral blood mononuclear cells were cultured in medium alone or in medium with 5 micrograms of PHA per ml for 48 h, and culture supernatants were collected for analysis. Cytokines selected for study were interleukin-10 (IL-10), gamma interferon (IFN-gamma), IL-2, and IL-4. All cytokine activities described were expressed as concentrations per 500,000 cells. We found that 48% (10 of 21) of the MS patients produced small but detectable levels of IL-10 in medium alone, compared with 26% (5 of 18) of the controls. We found that the MS patients produced significantly higher quantities of IL-10 protein than the controls in response to PHA (mean supernatant concentrations of IL-10 for patients and controls, 421 and 204 pg/ml, respectively [P < 0.05]). No significant differences were detected in the production of IL-2, IFN-gamma, and IL-4 between patients and controls in response to PHA, although patients appeared to display a trend toward decreased production of IFN-gamma.

Problem: The aim of this study was to characterize the phenotype of peritoneal lymphocyte and macrophage populations in mild versus severe endometriosis., Method: Using dual staining, antigen expression on peritoneal leukocytes from 24 women with endometriosis and 21 control patients was analyzed by flow cytometry., Results: All groups had CD4:CD8 ratios of 0.6, with subpopulations of CD8+ cells expressing cytotoxic marker S6F1. Mild and severe endometriosis patients had increased CD3/DR+ cells, relative to controls. Two populations of macrophages were identified by size in all groups. Mild endometriosis patients had increased percentages of small macrophages expressing CD14 and HLA DQ, compared to controls and severe disease patients. In severe disease patients, antigen expression on small macrophages did not differ from controls, but decreased percentages of large macrophages expressed CD14 relative to controls and mild disease patients., Conclusion: All women with endometriosis exhibit activated peritoneal lymphocytes, whereas macrophage expression of CD14 is differentially expressed as a function of disease stage. Alterations in the functional capacity of these cells may contribute to the pathophysiology of this disease.

A comprehensive peripheral blood immunophenotype analysis of 16 multiple sclerosis (MS) patients was performed by three-color flow cytometric analysis, and the results were compared with those for age-matched healthy controls. The cell subsets quantified included T cells (CD3+), B cells (CD19+), NK cells (CD56+), CD4+ and CD8+ T cells, cytotoxic (CD28+) and suppressor precursor (CD28-) CD8+ T cells, CD45RA+ and CD45RO+ T cells (CD4+ and CD8+), and CD5+ T and B cells. Analysis of MS patients' peripheral blood revealed essentially normal levels of total T, B, and NK cells. In agreement with results obtained by other investigators, it was found that MS patients had an increased CD4/CD8 ratio, primarily due to a decrease in CD8+ T cells. MS patients were found to have a significantly decreased level of suppressor precursor (CD28-) CD8+ T cells compared with that of controls but to have normal levels of cytotoxic (CD28+) CD8+ T cells. These data indicate that MS patients do not have a general decrease in CD8+ T cells but that they have a specific decrease in the suppressor precursor subset only and normal levels of cytotoxic CD8+ T cells. MS patients also had a significant increase in memory (CD45RO+) CD4+ T cells and displayed a trend towards a decrease in naive (CD45RA+) T cells in the peripheral blood.

Thioglycolate-elicited murine macrophages from genetically susceptible A/J mice activated with lipopolysaccharide (LPS) and infected with Legionella pneumophila in vitro evince marked inhibition of intracellular growth of this bacterium. The mechanism of inhibition by LPS-activated macrophages in terms of replication of this intracellular pathogen is unclear. LPS activation of murine macrophages induced a downshift in transferrin receptor (TfR) expression and reduction in cellular iron content, and this was correlated with augmented intracellular growth of Legionella in the cells. When LPS-stimulated macrophages were first saturated with iron, partial reversion of L. pneumophila growth restriction was observed. However, an excess of exogenous L-tryptophan (Trp) did not reverse this growth inhibition, nor did supplementation of the macrophage culture medium with both iron and Trp. The antilegionella activity of the macrophages induced by LPS activation was independent of reactive oxygen intermediates (ROI), since the scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on growth restriction. Likewise, notwithstanding the ability of LPS-activated macrophages to synthesize reactive nitrogen intermediates (RNI), which was inhibited by L-arginine analogs NG-monomethyl-L-arginine and L-aminoguanidine), as well as by incubation in arginine-free medium, their ability to inhibit the intracellular replication of L. pneumophila was not affected. Thus, we conclude that LPS-activated macrophages inhibit the intracellular growth of L. pneumophila partially by iron-dependent, Trp-independent, and ROI- and RNI-independent mechanisms. We also suggest that additional unknown mechanisms are involved, since complete reversion was not obtained.

We have investigated the modulation of iron in two populations of macrophages which differ in susceptibility to Legionella pneumophila intracellular proliferation. Previously, we reported that thioglycolate-elicited peritoneal macrophages obtained from the inbred A/J mouse strain readily support the intracellular growth of L. pneumophila, while resident macrophages from the same strain do not. In this study, we show that A/J elicited macrophages exhibit markedly higher expression of transferrin receptor and intracellular iron content than A/J resident macrophages. Furthermore, apotransferrin and desferrioxamine inhibited the intracellular proliferation of L. pneumophila in elicited macrophages, and this suppression was reversed by the additions of Fe-transferrin or ferric nitrilotriacetate. Fe-transferrin and ferric nitrilotriacetate did not further increase the intracellular proliferation of L. pneumophila in thioglycolate-elicited macrophages. However, ferric citrate and ferric nitrilotriacetate stimulated in a dose-dependent manner the growth of L. pneumophila in resident macrophages. Furthermore, equimolar concentrations of desferrioxamine reversed the stimulatory effect of iron in these resident cells. These data provide evidence supporting the hypothesis that differences in susceptibility to L. pneumophila growth between permissive elicited macrophages and nonpermissive resident macrophages from the A/J mouse strain are due to intracellular availability of iron.