Monocytes and macrophages synthesize and secrete thrombospondin

Thrombospondin, one of the major glycoproteins released from alpha- granules of thrombin-stimulated platelets, is a disulfide-linked trimer of 160,000-dalton subunits. Cultured human monocytes secreted thrombospondin (determined by an enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (1.45 micrograms/10(6) cells/24 hr); secretion was totally blocked by cycloheximide (1 microgram/mL). 35S-thrombospondin was isolated from 35S-methionine-labeled human monocyte postculture medium with rabbit polyclonal anti-thrombospondin coupled to protein A-Sepharose. The immunoisolated 35S-thrombospondin migrated in sodium dodecyl sulfate- polyacrylamide gels after reduction with a molecular weight of 159,000. Similar results were obtained using mouse resident peritoneal macrophages. Elicited peritoneal macrophages harvested from mice pretreated with endotoxin, casein, or thioglycollate secreted much less thrombospondin than did resident macrophages harvested from control mice. Thus, monocytes and macrophages from two different species synthesize and secrete thrombospondin, and the rate of synthesis of thrombospondin appears to depend on the state of activation of the cells.     

Antigenic cross-reactivity between the human whipworm, Trichuris trichiura, and the mouse trichuroids Trichuris muris and Trichinella spiralis

Cross-reactivity was demonstrated between circulating antibodies from Trichuris trichiura-infected humans and T. muris-infected mice for heterologous antigen preparations. Mouse immune sera raised against excretory/secretory (E/S) products and anterior end homogenate from adult T. muris showed marked affinity for T. trichiura adult homogenate in ELISA, and 35S-methionine-labelled adult T. muris E/S products were precipitated by T. trichiura infection sera. Monoclonal antibodies recognizing a 48 kD Trichinella spiralis muscle larval beta-stichocyte granule antigen also showed avidity for T. trichiura adult homogenate in ELISA, as did serum from mice with patent T. muris worm burdens. This cross-reactivity is thought to result from shared stichocyte antigens and the relevance of this observation is discussed.     

CHEK2*1100delC homozygosity in the Netherlands—prevalence and risk of breast and lung cancer

The 1100delC mutation in the CHEK2 gene has a carrier frequency of up to 1.5% in individuals from North-West Europe. Women heterozygous for 1100delC have an increased breast cancer risk (odds ratio 2.7). To explore the prevalence and clinical consequences of 1100delC homozygosity in the Netherlands, we genotyped a sporadic breast cancer hospital-based cohort, a group of non-BRCA1/2 breast cancer families, and breast tumors from a tumor tissue bank. Three 1100delC homozygous patients were found in the cohort of 1434 sporadic breast cancer patients, suggesting an increased breast cancer risk for 1100delC homozygotes (odds ratio 3.4, 95% confidence interval 0.4–32.6, P=0.3). Another 1100delC homozygote was found in 592 individuals from 108 non-BRCA1/2 breast cancer families, and two more were found after testing 1706 breast tumors and confirming homozygosity on their wild-type DNA. Follow-up data was available for five homozygous patients, and remarkably, three of them had developed contralateral breast cancer. A possible relationship between 1100delC and lung cancer risk was investigated in 457 unrelated lung cancer patients but could not be confirmed. Due to the small number of 1100delC homozygotes identified, the breast cancer risk estimate associated with this genotype had limited accuracy but is probably higher than the risk in heterozygous females. Screening for CHEK2 1100delC could be beneficial in countries with a relatively high allele frequency.     

Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections

Acute HIV infection is characterized by massive loss of CD4 T cells from the gastrointestinal (GI) tract. Th17 cells are critical in the defense against microbes, particularly at mucosal surfaces. Here we analyzed Th17 cells in the blood, GI tract, and broncheoalveolar lavage of HIV-infected and uninfected humans, and SIV-infected and uninfected sooty mangabeys. We found that (1) human Th17 cells are specific for extracellular bacterial and fungal antigens, but not common viral antigens; (2) Th17 cells are infected by HIV in vivo, but not preferentially so; (3) CD4 T cells in blood of HIV-infected patients are skewed away from a Th17 phenotype toward a Th1 phenotype with cellular maturation; (4) there is significant loss of Th17 cells in the GI tract of HIV-infected patients; (5) Th17 cells are not preferentially lost from the broncheoalveolar lavage of HIV-infected patients; and (6) SIV-infected sooty mangabeys maintain healthy frequencies of Th17 cells in the blood and GI tract. These observations further elucidate the immunodeficiency of HIV disease and may provide a mechanistic basis for the mucosal barrier breakdown that characterizes HIV infection. Finally, these data may help account for the nonprogressive nature of nonpathogenic SIV infection in sooty mangabeys.     

The pharmacokinetic profile of raltegravir-containing antiretroviral therapy in HIV-infected individuals over 60 years of age

Background:

Antiretroviral safety and efficacy and may differ in older versus younger HIV-infected patients. The objective of this study was to assess the pharmacokinetic (PK) profile in older HIV-infected subjects (>60?years) switching combination antiretroviral therapy (cART) to a raltegravir (RAL) containing regimen.

Methods:

Nineteen HIV-infected patients over 60?years of age on effective cART (HIV-RNA Results:

In HIV-infected subjects over the age of 60 (mean?±?SD age: 66?±?3.4?years, n?=?19) switching to a RAL containing regimen, we observed no safety concerns, no plasma virological rebounds, and no differences in RAL apparent oral clearance when compared to younger HIV-infected populations (mean?±?SD age: 41?±?9.2?years, n?=?38) based on population pharmacokinetic analysis. After 24?weeks of study therapy a decline in cognitive function was observed [change in (SD) global score of (0.91 (1.3), P?=?0.018].

Conclusions:

No significant changes in RAL exposure associated with age were observed.     

Systemic use of non‐biologic agents in orofacial diseases: other immunomodulatory agents

Systemic non‐biologic agents have long been in clinical use in medicine – often with considerable efficacy, albeit with some adverse effects – as with all medications. With the advent of biologic agents, all of which currently are restricted to systemic use, there is a growing need to ensure which agents have the better therapeutic ratio. The non‐biologic agents (NBAs) include a range of agents, most importantly the corticosteroids (steroids). Previous articles by us in this series have discussed systemic use of corticosteroids and purine synthesis inhibitors; the other immunomodulating agents (calcineurin inhibitors, thalidomide, dapsone, colchicine and cyclophosphamide) are reviewed in this final article.     

Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages

Many significant bacterial pathogens have evolved virulence mechanisms to evade degradation and exposure to reactive oxygen (ROS) and reactive nitrogen species (RNS), allowing them to survive and replicate inside their hosts. Due to the highly reactive and short-lived nature of ROS and RNS, combined with limitations of conventional detection agents, the mechanisms underlying these evasion strategies remain poorly understood. In this study, we describe a system that uses redox-sensitive GFP to nondisruptively measure real-time fluctuations in the intrabacterial redox environment. Using this system coupled with high-throughput microscopy, we report the intrabacterial redox dynamics of Salmonella enterica Typhimurium (S. Typhimurium) residing inside macrophages. We found that the bacterial SPI-2 type III secretion system is required for ROS evasion strategies and this evasion relies on an intact Salmonella-containing vacuole (SCV) within which the bacteria reside during infection. Additionally, we found that cytosolic bacteria that escape the SCV experience increased redox stress in human and murine macrophages. These results highlight the existence of specialized evasion strategies used by intracellular pathogens that either reside inside a vacuole or “escape” into the cytosol. Taken together, the use of redox-sensitive GFP inside Salmonella significantly advances our understanding of ROS and RNS evasion strategies during infection. This technology can also be applied to measuring bacterial oxidative and nitrosative stress dynamics under different conditions in a wide variety of bacteria.A central mechanism of the innate immune response to defend against pathogens is the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by specialized phagocytic immune cells (1). Macrophages and neutrophils generate ROS after detection of pathogen-associated molecular patterns (PAMPs) through the NADPH oxidase complex, whereas RNS is produced by inducible nitric oxide synthase (iNOS), which generates NO• through the conversion of l-arginine and oxygen (1–3).To survive, replicate, and disseminate throughout the body, bacterial pathogens—especially those that reside in intracellular niches—must overcome the antimicrobial oxidative and nitrosative burst (1). General ROS and RNS enzymes such as catalases, peroxidases, superoxide dismutases, and DNA repair enzymes are used by most bacteria to survive exposure to ROS and RNS (4). In addition to these general defenses, many intracellular pathogens have evolved specific evasion strategies that allow them to live inside their host cells. For example, the intracellular pathogens Shigella flexneri and Listeria monocytogenes, which cause shigellosis and listeriosis, respectively, “escape” the phagolysosome to proliferate within the cytosol of macrophages. In contrast, the Gram-negative pathogen Salmonella enterica subsp. Typhimurium (S. Typhimurium), which is a major cause of gastroenteritis and some systemic diseases, remains inside a specific Salmonella-containing vacuole (SCV), where it injects bacterial effector proteins directly into the host cell through a type III secretion system (T3SS). A specific set of type III effectors associated with a Salmonella pathogenicity island (SPI), known as SPI-2 effectors, have been implicated in ROS and RNS evasion strategies (5, 6); however, the relationship between SPI-2 and oxidative stress evasion is a contentious topic after a recent study concluded that the contribution of SPI-2 to Salmonella pathogenesis is unrelated to its interaction with oxidative stress (7). Inadequate analytic tools for directly measuring ROS/RNS and rapid fluctuations due to the short-lived nature of ROS/RNS have also contributed to poor reproducibility in the studies of ROS and RNS evasion strategies (1, 8, 9).Herein, we describe a previously unidentified use of a redox-sensitive GFP (roGFP2) that has been engineered to form a reversible disulfide bond upon oxidation or S-nitrosylation of specific cysteines. Formation of the disulfide bond leads to a slight shift in its conformation (Fig. S1A) resulting in two isoforms (roGFP2_ox and roGFP2_red) of roGFP2. These isoforms have distinct excitation spectra with specific fluorescence signals after excitation at 405 and 480 nm, respectively (10). Consequently, the 405/480-nm ratio can be used as a measure for the roGFP2_ox/roGFP2_red ratio, which corresponds with the intrabacterial redox potential (11). Because roGFP2 reports the redox potential by ratiometric analysis, this system excludes variations due to differences in roGFP2 concentrations. Until now, redox-sensitive GFP has almost exclusively been used in eukaryotes (12). In this study, we used roGFP2 to measure the intrabacterial redox potential in bacteria (Salmonella) after challenges with exogenous oxidative agents and during infection of HeLa cells (epithelial), THP-1 cells (monocytic), and bone marrow-derived macrophages (BMDMs) from mice. Finally, with the use of high-throughput microscopy, we demonstrated the involvement of the SPI-2 T3SS in ROS evasion strategies, as well as the requirement for an intact SCV to evade ROS and RNS inside macrophages.     

Occupational Chronic Obstructive Pulmonary Disease in a Danish Population-Based Study

The aim was to explore the impact of occupation on chronic obstructive pulmonary disease (COPD) in a cross-sectional population-based study among subjects aged 45 to 84 years. In a stratified sampling 89 general practitioners practices (GPP) in Denmark recruited 3106 males and 1636 females through the Danish Civil Registration System. COPD was defined by spirometry by the 2.5^th-centile Lower Limit of Normal of FEV₁ and FEV₁/FVC. Information about smoking, occupational exposure and the respective occupations were obtained from questionnaires. Occupations followed the Danish adaptation of The International Standard Classification of Occupations, revision 1988 (DISCO-88). Exposure to vapour, gas, dust (organic and inorganic), and fume (VGDF) in each occupation (yes/no) was evaluated by two independent specialist in occupational medicine. Exposures were divided in no, low, medium, and high exposure as 0, <5, 5–14, and ≥ 15 years in the job, respectively. Data was analysed by a mixed random effect logistic regression model. The age-standardised COPD study prevalence was 5.0%. Of 372 DISCO-88 codes 72 were identified with relevant exposure to VGDF. 46% of the participants reported at least one occupation with VGDF exposure. Adjusted for smoking, age, sex, and GPP a dose-dependent association of COPD was found among workers in jobs with high organic dust exposure, with OR 1.56 (95% CI 1.09–2.24). Restricted to agriculture the OR was 1.59 (95% CI: 1.08–2.33). No association was observed for workers in jobs with inorganic dust, fume/gas, or vapour exposures. In summary, occupational organic dust exposure was associated to the prevalence of COPD.