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Understanding the biology of the tuberculosis pathogen during dormant asymptomatic infection, called latent tuberculosis is crucial to decipher a resilient therapeutic strategy for the disease. Recent discoveries exhibiting presence of pathogen's DNA and bacilli in mesenchymal stem cells (MSCs) of human and mouse despite completion of antitubercular therapy, indicates that these specific cells could be one of the niches for dormant Mycobacterium tuberculosis in humans. To determine if in vitro infection of human MSCs could recapitulate the in vivo characteristics of dormant M. tuberculosis, we examined survival, phenotype, and drug susceptibility of the pathogen in MSCs. When a very low multiplicity of infection (1:1) was used, M. tuberculosis could survive in human bone marrow derived MSCs for more than 22 days without any growth. At this low level of infection, the pathogen did not cause any noticeable host cell death. During the later phase of infection, MSC-residing M. tuberculosis exhibited increased expression of HspX (a 16-kDa alpha-crystallin homolog) with a concurrent increase in tolerance to the frontline antitubercular drugs Rifampin and isoniazid. These results present a human MSC-based intracelllular model of M. tuberculosis infection to dissect the mechanisms through which the pathogen acquires and maintains dormancy in the host., Competing Interests: Declaration of Competing Interest Authors declare no financial or competing interest with any organization or personnel., (Copyright © 2020 Institut Pasteur. All rights reserved.)

Murine models of Mycobacterium tuberculosis (Mtb) infection demonstrate progression of M1-like (proinflammatory) and M2-like (anti-inflammatory) macrophage morphology following primary granuloma formation. The Mtb cell wall cording factor, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant and useful molecule for modeling early macrophage-mediated events during establishment of the tuberculosis-induced granuloma pathogenesis. Here, it is shown that TDM is a major driver of the early M1-like macrophage response as seen during initiation of the granulomas of primary pathology. Proinflammatory cytokines tumor necrosis factor-α, IL-1β, IL-6, and IL-12p40 are produced in lung tissue after administration of TDM to mice. Furthermore, CD11b + CD45 + macrophages with a high surface expression of the M1-like markers CD38 and CD86 were found present in regions of pathology in lungs of mice at 7 days post-TDM introduction. Conversely, only low phenotypic marker expression of M2-like markers CD206 and EGR-2 were present on macrophages. These findings suggest that TDM plays a role in establishment of the M1-like shift in the microenvironment during primary tuberculosis., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The immune system responds to tuberculosis (TB) infection by forming granulomas. However, subsequent immune-mediated destruction of lung tissue is a cause of significant morbidity and contributes to disease transmission. Lactoferrin, an iron-binding glycoprotein, has demonstrated immunomodulatory properties that decrease tissue destruction and promote T(H)1 immune responses, both of which are essential for controlling TB infection. The cord factor trehalose 6,6'-dimycolate (TDM) model of granuloma formation mimics many aspects of TB infection with a similar histopathology accompanied by proinflammatory cytokine production. C57BL/6 mice were injected intravenously with TDM. A subset of mice was given 1 mg of bovine lactoferrin 24 h post-TDM challenge. Lung tissue was analyzed for histological response and for the production of proinflammatory mediators. C57BL/6 mice demonstrated a granuloma formation that correlated with an increased production of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α,) IL-12p40, interferon-gamma (IFN-γ), and IL-10 protein. Mice treated with lactoferrin postchallenge had significantly fewer and smaller granulomas compared with those given TDM alone. Proinflammatory and T(H)1 cytokines essential to the control of mycobacterial infections, such as TNF-α and IFN-γ, were not significantly different in mice treated with lactoferrin. Furthermore, the anti-inflammatory cytokines IL-10 and transforming growth factor-β were increased. A potential mechanism for decreased tissue damage observed in the lactoferrin-treated mice is proposed. Because of its influence to modulate immune responses, lactoferrin may be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection., (Copyright © 2010 Mosby, Inc. All rights reserved.)

Trehalose 6,6'-dimycolate (TDM) is the most abundant lipid extracted from Mycobacterium tuberculosis (MTB). TDM promotes MTB survival by decreasing phagosomal acidification and phagolysosomal fusion in macrophages. Delipidation of MTB using petroleum ether removes TDM and decreases MTB survival within host cells. TDM reconstituted onto MTB restores its virulent wild-type characteristics. We investigated the role of TDM in regulating surface marker expression in MTB-infected macrophages. Macrophages were infected with wild-type, delipidated, and TDM-reconstituted MTB for 24h and measured for changes in surface marker expression. TDM on MTB was found to specifically target MHCII, CD1d, CD40, CD80 and CD86. Both wild-type and TDM-reconstituted MTB suppressed or induced no change in expression of these surface markers, whereas delipidated MTB increased expression of the same markers. MTB-infected macrophages were also overlaid with MHCII-restricted T cell hybridomas which recognize Antigen 85B. Macrophages infected by wild-type and TDM-reconstituted MTB did not present antigen as well as delipidated MTB-infected macrophages. The evidence shown furthers supports the notion that TDM present on MTB promotes its survival and persistence in host macrophages.

Objective: The purpose of our study was to determine whether physician attire played a role in patient satisfaction after a new obstetrician/gynecologist encounter., Study Design: Over a 3-month period, 20 physicians were randomly assigned to dress in business attire, casual clothing, or scrub suit each week. One thousand one hundred sixteen patients who had an office visit with a new obstetrician/gynecologist for at least 10 minutes completed a satisfaction survey, which assessed patient comfort as well as perception of the competency and professionalism of the physician. Patients were blinded to the physician attire manipulation., Results: There was no difference in the mean overall satisfaction score among the 3 physician attire groups. No differences existed in satisfaction scores when analyzed by individual survey item or by demographic factors, after controlling for attire., Conclusion: Patients are equally satisfied with physicians who dress in business attire, casual clothing, or scrub suit.

Trehalose 6,6'-dimycolate (TDM) is the most abundant, most granulomagenic, and most toxic lipid extractable from the surface of virulent Mycobacterium tuberculosis (MTB). We further examined its toxicity, which requires activation by oily surfaces. Injections of MTB and/or TDM into sensitized mice induced caseating granulomas that centered on oil droplets. If large doses of MTB were injected in saline, caseating granulomas developed in adipose tissue, but MTB with surface TDM removed induced only acute inflammation that did not persist. Variations in protocols produced several variants of caseating granulomas, each with characteristics of human tuberculosis. In each instance, MTB were localized in fat cells or oil drops during initiation of caseating granulomas suggesting that necrosis was caused by activation of the toxicity of TDM toxicity. Evidence extending these findings to the lung was derived from the observation that in sensitized mice, as in humans, tuberculosis development stimulates accumulation of lipid selectively in alveoli. MTB preferentially associated with lipid droplets in developing necrotic foci in late-stage murine tuberculosis. This supports the hypothesis that pulmonary tuberculosis sequesters MTB in a protected environment that accumulates lipid until it is able to activate the toxicity of TDM and initiate necrosis that results in caseating granulomas.

Objective: The objective of this study was to evaluate the willingness of Philadelphia obstetrics and gynecology residents to participate in three abortion procedures for various fetal conditions., Methods: Anonymous questionnaires were distributed to 310 residents from 18 programs. The survey asked the residents whether they would participate in first trimester dilatation and evacuation (D&E), second trimester prostaglandin induction or second trimester D&E for the following conditions: lethal fetal anomaly, nonlethal anomaly with certain long-term functional consequences, possible long-term functional consequences, little or no long-term functional consequences and elective abortion of a normal fetus., Results: Of the 148 respondents, the percentage of residents who would participate in a second trimester D&E for each fetal condition was significantly lower than that for a first trimester D&E (p < or = .001). Additionally, for each abortion procedure, the participation rates consistently fell for lesser degrees of fetal severity. Participation was significantly associated with preferences regarding abortion legislation and personal abortion stance., Conclusion: Resident attitudes regarding abortion participation were related to severity of the fetal condition, gestational age and procedure type.

Tobramycin is an aminoglycoside used in the treatment of infection against gram-negative bacteria. Tobramycin cannot be delivered orally probably due to efflux of drug by a P-glycoprotein pump in the brush border of the small intestine. In this report we demonstrate oral delivery of tobramycin in FVB mice using CRL-1605 copolymer as a vehicle. This copolymer is known to inhibit P-glycoprotein. Two different doses of tobramycin (25 mg/kg and 200 mg/kg) were used. The concentration of CRL-1605 copolymer was 132 mg/kg. The liquid formulation was fed to mice by gavage and serum tobramycin concentrations were measured after one and two hours using the fluorescence polarization immunoassay. We observed significant increases in serum tobramycin concentrations when the drug was delivered orally with the copolymer compared to when the drug was delivered alone. We also performed a bioassay using Bacillus subtilis to confirm antibacterial effect of tobramycin in mice sera. This was to ensure that tobramycin did not undergo structural change during oral absorption when delivered in the copolymer vehicle. We observed minimal inhibition in growth of Bacillus subtilis in sera obtained from mice fed with tobramycin alone. In contrast, we observed almost complete inhibition of growth (most specimens) in sera obtained from mice fed with tobramycin in the presence of CRL-1605 copolymer. We conclude that tobramycin delivered orally in mice using copolymer 1605 is also bioactive.

Amikacin is an aminoglycoside which is used in the treatment of infection from gram negative bacteria. Amikacin is also used synergistically with penicillin against gram positive cocci. Amikacin cannot be delivered orally probably due to efflux of drug by P-glycoprotein pump in the brush border of intestine. We studied the possibility of delivering amikacin orally in mice using a copolymer (CRL-1605) as a vehicle. This copolymer inhibits P-glycoprotein pump. Two different doses of amikacin were used (500 mg/kg and 100 mg/kg). The concentration of polymer used was 132 mg/kg. The liquid formulation was fed to mice by gavage and serum amikacin concentrations were estimated after one hour and two hours using fluorescence polarization immunoassay. We observed a two fold increase in serum amikacin concentration when amikacin was orally delivered in the presence of CRL-1605 compared to controls (amikacin alone). We conclude that gastrointestinal absorption of amikacin is significantly increased in the presence of CRL-1605 in mice.

Because of its high sensitivity, bioluminescence (BL) is an excellent alternative to radioactive quantitation of cytokine RT-PCR-derived products. BL also allows detection of amplicons at cycle numbers not normally detectable using radioactivity. No direct comparisons between these two methods have been made. In this study, the sensitivities of BL using recombinant aequorin, a flash-type luminescent tag capable of detecting signal to attomolar (10(-18) M) levels and radio imaging (RI) were directly compared. In addition, the application of BL for detecting cytokine message from biologic samples was examined. BL was 30- to 60-fold more sensitive than RI in detecting human IL-2 and CD3delta amplicons. This difference was particularly found during low cycle PCR, but was less at higher cycle numbers. The ability of BL to detect differences in cytokine message in stimulated and unstimulated human peripheral blood mononuclear cells was also evaluated. Using linear regression analysis, we observed up to 5,000-fold increases in RT-PCR amplified-mRNA in stimulated cells for IL-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10 and GM-CSF compared to unstimulated cells. Changes in CD3delta, TNF alpha or IL-12 were not observed or quantitated. We present a novel aequorin-based application of bioluminescent technology to directly quantitate RT-PCR amplicons and to investigate the induction of human cytokine expression. Significant advantages of this sensitive bioluminescent method compared with radioactive methods are its abilities to quantitate amplicons in a PCR cycle range where linear detection is most robust and to analyze products in an automated, open-architecture microtiter plate format.

The induction of nitric oxide in human monocytes during mycobacterial infection has been a controversial issue. This study describes a comparative evaluation of the colorimetric and fluorometric methods for the detection of NO in response to Mycobacterium tuberculosis (MTB) infection in human peripheral blood-derived monocytes (PBM) and in U937, a human monocyte-derived cell line. MTB was grown in monocyte cultures in vitro for 7 or 10 days. RPMI 1640 medium, without antibiotics and supplemented with L-arginine, Hepes, 5% human AB serum, and tetrahydrobiopterin was used to support monocyte growth. As early as 72 h after infection, soluble nitrite was detectable in the medium using the fluorometric assay with diaminonaphthalene (DAN). Early induction of NO correlated with an increase in the levels of iNOS mRNA as quantitated by RT-PCR. NO levels increased progressively up to day 10 (PBM) or day 7 (U937), when 150-200 nM/10(6) cells of soluble nitrite accumulated in cultures, as measured by DAN. Furthermore, monocytes stained positively for human iNOS protein and peroxynitrite after infection with MTB. The induction of NO by MTB was inhibited by four different inhibitors of iNOS enzyme including N-monomethylarginine. Inhibition of NO resulted in the enhancement of the intracellular growth of two of five clinical isolates of MTB. NO released from a donor (S-nitroso-N-penicillamine) also had a direct bacteriostatic effect on the same isolates in broth cultures. MTB strains thus showed a differential susceptibility to intracellular and extracellular NO. In most of these assays, the Greiss reagent was limited by its sensitivity and remained negative for soluble nitrite throughout the 7-10 days of incubation. Thus, the colorimetric method, which is widely used, may give false-negative results in NO assays. This report also demonstrates for the first time that MTB induces mRNA for iNOS, iNOS protein, NO, and peroxynitrite in human monocyte/macrophage cultures.

Nonionic triblock copolymers are relatively nontoxic adjuvants that induce high-titer, long-lasting antibody responses. We have previously shown that these adjuvants also induce cell-mediated immunity including lymphokine production by CD4(+) T cells and cytolytic responses by CD8(+) T cells. These copolymers are thought to modulate hydrophobic adhesive interactions between antigens (Ag) and lymphoid cells. We sought to test the hypothesis that copolymers facilitate uptake of exogenous Ag by antigen-presenting cells (APC) using an in vitro model system. Our data show that nonionic triblock copolymers enhanced presentation of soluble ovalbumin (OVA) to the major histocompatibility complex (MHC) class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells, respectively. Presentation of OVA via the class I pathway was enhanced by copolymers in both phagocytic and nonphagocytic APC. However, copolymers did not enhance binding of peptides to the MHC molecules on APC, presentation of endogenously synthesized Ag, or presentation of exogenous Ag delivered by electroporation. These results provide additional evidence that these nonionic triblock copolymers can serve as powerful adjuvants for augmenting both humoral and cell-mediated immunity to protein Ag.

The hypothesis of this study was that D dimer, a specific degradation product of cross-linked fibrin, would be increased in the bronchoalveolar lavage (BAL) fluids of patients with sarcoidosis and that it would be related to other BAL parameters of disease activity. Eight of 10 sarcoidosis patients but none of 18 healthy volunteers had detectable BAL D dimer by enzyme immunoassay. Autoradiography revealed the presence of fibrinogen and D dimer in the BAL fluids from sarcoidosis patients. Bronchoalveolar lavage D dimer levels in sarcoidosis patients correlated with total BAL cells per milliliter, lymphocytes per milliliter, and total protein level, but not macrophages per milliliter. The D dimer in the BAL fluids from sarcoidosis patients did not correlate with D dimer in the blood. Our findings indicate that BAL D dimer parallels directly the lymphocytic alveolitis that characterizes pulmonary sarcoidosis.

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

We have developed a polyacrylamide gradient gel electrophoretic method to quantitate apo-, mono-, and diferric transferrin based upon differences in their molecular size. Purified transferrin saturated to different extents (3% to 98%) with iron showed proportions of the three forms as predicted from an approximately random distribution of iron between the two metal-binding sites. The iron distributions in sera of 14 normal individuals similarly correlated with the predicted values. In contrast, 22 of 43 patients with diseases associated with abnormalities in iron or transferrin metabolism had a disproportionate increase in monoferric transferrin. This abnormality occurred in seven of nine patients who had received bone marrow transplants, seven of 14 with chronic liver disease, and eight of nine menstruating women with probable iron deficiency anemia. Interestingly, 11 patients with malabsorption or chronic renal disease had normal iron distributions. The finding of abnormal distributions of iron on transferrin suggests that gradient gel analysis may be a useful tool for studying the physiologic mechanisms controlling iron utilization.

We have developed an enzyme-linked immunosorbent assay (ELISA) to measure antibody production in cell suspensions using highly sensitive avidin-biotinylated peroxidase (ABC) reagents. The cells are serially diluted in media and placed directly on a standard antigen-coated microtiter plate for 6 h, and the plate is then washed and processed to determine the bound antibody. The amount of antibody detected is reduced by puromycin indicating in vitro synthesis. Standard errors of the means of optical density readings of replicate samples are less than 10%. The background readings and readings obtained using non-immune cells are negligible, demonstrating no significant contamination by endogenous peroxidases.

Axillary lymph nodes obtained from 37 patients with cancer of the breast were used in a microcytotoxicity assay against a human mammary cancer cell line, ALAB. Cell suspensions made from 60 individual lymph nodes and 51 lymph node pools were tested. Each lymph node was graded for the extent of the histologic changes of sinus histiocytosis (SH), paracortical hyperplasia (PCH), and germinal center hyperplasia (GCH). High levels of cytotoxicity correlated significantly with the presence of SH and PCH. When multiple lymph nodes of individual patients were pooled and tested, a high intensity of GCH correlated with a low degree of cytototoxicity. The cytotoxic activity of pooled lymph nodes from different axillary regions was studied in 20 patients. In 7 of these patients, low axillary lymph node cells were more cytotoxic than high axillary node cells. The reverse was found for 7 patients, and there was no difference in cytotoxicity between axillary regions in 6. Cytotoxic lymph node pools had high SH and low GCH whether they were obtained from the low or high region. Noncytotoxic lymph node pools from the high axillary region had a low intensity of all three histologic reactions. In contrast, noncytotoxic lymph node pools from the low axillary regions had high intensities of SH, PCH, and GCH. These results suggest that germinal center hyperplasia in the lymph nodes nearest an advancing tumor is associated with a local suppression of cytotoxic cell activity.