Symbolic and anticipated HIV stigma are associated with mental health and education in South Africa.

HIV stigma continues to act as a barrier to HIV care in South Africa, necessitating further research on the intersections of socioeconomic factors and the anticipation and expression of stigma surrounding HIV. We measured the prevalence of HIV-related stigma and evaluated factors associated with symbolic and anticipated stigma in Umlazi Township, South Africa from 2013 to 2019, using a validated HIV stigma scale, before undergoing HIV testing. Among 7,724 people evaluated, 1,318 (16.9%) reported symbolic stigma and 2,396 (30.8%) anticipated HIV stigma. Prevalence of symbolic and anticipated stigma were significantly more common among both women and people living with HIV, compared to men and those who tested negative for HIV. In multivariable analyses, higher education and depressive symptoms were the strongest correlates with both symbolic stigma and anticipated stigma. Younger age, not being married, and having a partner who was not living with HIV appeared to be important correlates with anticipated stigma, but not symbolic stigma. Overall, the anticipation of experiencing stigma because of infection with HIV continues to be an important factor in the testing and management of HIV.

Vasculitis Presenting as Autonomic Failure.

INTRODUCTION: Granulomatosis with polyangiitis (GPA) is a vasculitic process that can cause neurological dysfunction in addition to characteristic sinus and pulmonary manifestations. This case report highlights the spectrum of nervous system manifestations and includes rarely reported autonomic and pituitary involvement. CASE REPORT: A 62-year-old woman presented with orthostatic intolerance, tachycardia, dry mouth, and temperature sensitivity; subsequent autonomic reflex study demonstrated widespread postganglionic sympathetic sudomotor, cardiovagal, and cardiovascular adrenergic impairment reflective of autonomic neuropathy and overall autonomic failure. Additional progressive symptoms included dysarthria, dysphagia, bilateral hearing loss, voice hoarseness, and right-sided facial numbness with multiple cranial neuropathies identified on neurological examination. The diagnosis of central diabetes insipidus was also confirmed. Pachymeningitis was present on brain magnetic resonance imaging. Pathologic review of the dural biopsy specimen revealed necrotizing granulomatous vasculitis consistent with GPA. She was treated with intravenous methylprednisolone and rituximab. Over the next 2 months, she had near-complete resolution of her symptoms with normalization on repeat autonomic testing. CONCLUSIONS: This is a unique GPA case presenting with autonomic failure and pituitary dysfunction with conclusive objective findings of autonomic dysfunction. Autonomic dysfunction and other disease manifestations were responsive to immunosuppressive therapy.

Clinical implications of synovial fluid specimen handling for crystal associated arthritides: A systematic review.

AIM: To identify the appropriate methods of synovial fluid (SF) specimen storage, manipulation and handling for crystal associated arthritides (CAA) diagnosis. METHOD: A systematic literature review was conducted using 5 medical databases to identify diagnostic studies assessing SF specimen handling for calcium pyrophosphate (CPP) and monosodium urate (MSU) crystals identification. All included studies were rated for quality using the Quality Assessment of Diagnostic Accuracy Studies 2. RESULTS: Fifteen studies, including 2 non-English language manuscripts, were included. Eight studies examined both types of crystals, while 3 studies examined CPP and 4 studies examined MSU crystals only. Overall, MSU crystals were more stable over time compared to CPP crystals. MSU stability was generally independent of time, preservative and temperature. CPP crystals deteriorated with time and were more stable if refrigerated. Ethylenediaminetetraacetic acid (EDTA) was a suitable preservative. Re-examining an initially negative SF sample at 24 hours facilitated detection of additional cases. Very few studies had an overall low risk of bias and applicability. CONCLUSION: Monosodium urate crystals remain stable over time independent of storage time, temperature and preservative. CPP crystals are mostly stable for 24-48 hours but can deteriorate with time. Overall, SF crystal examination should ideally be done within 24-48 hours. They may be stored at room temperature without any preservative. Otherwise, refrigeration (4°C/39°F) and EDTA preservation is reasonable. Stored SF re-examination, at 24 hours, helps identify a small number of additional MSU and CPP cases. Centrifugation techniques allow better and easier crystal identification, particularly CPP. Most studies were of unclear or low quality.

Efficacy of mitoxantrone-based salvage therapies in relapsed or refractory acute myeloid leukemia in the Mayo Clinic Cancer Center: Analysis of survival after 'CLAG-M' vs. 'MEC'.

Salvage therapy regimens for refractory and relapsed AML include mitoxantrone, etoposide, and cytarabine (MEC) and cladribine, cytarabine, filgrastim, and mitoxantrone (CLAG-M). We analyzed patients receiving either CLAG-M or MEC as salvage therapy for RR-AML between 09/01/2009-12/31/2017. Of 150 patients with RR-AML, 34 patients received CLAG-M and 116 MEC. CR/CRi rates for CLAG-M and MEC were 61.3 % (19/31) and 55.6 % (60/108). Median OS was 9.5 months for CLAG-M and 10.0 months for MEC (HR = 0.88,95 %CI = 0.54-1.41,p = 0.59). 76 patients proceeded to ASCT following salvage therapy. Median OS after ASCT was 13.0 months for CLAG-M and 31.0 months for MEC (HR = 1.76,95 %CI = 0.87-3.56,p = 0.12). Among those with late relapse and ASCT, median OS was 9.0 and 48.0 months for CLAG-M and MEC, respectively (HR = 17.6,95 %CI = 1.57-198,p < 0.001). There were no significant differences in outcome between CLAG-M vs. MEC regardless of transplant status. There was a significant improvement in survival in patients with late relapse treated with MEC who proceeded to ASCT.

Aberrant regulation of epigenetic modifiers contributes to the pathogenesis in patients with selenoprotein N-related myopathies.

Congenital myopathies are early onset, slowly progressive neuromuscular disorders of variable severity. They are genetically and phenotypically heterogeneous and caused by pathogenic variants in several genes. Multi-minicore Disease, one of the more common congenital myopathies, is frequently caused by recessive variants in either SELENON, encoding the endoplasmic reticulum glycoprotein selenoprotein N or RYR1, encoding a protein involved in calcium homeostasis and excitation-contraction coupling. The mechanism by which recessive SELENON variants cause Multiminicore disease (MmD) is unclear. Here, we extensively investigated muscle physiological, biochemical and epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, to understand the pathomechanism of MmD. We identified biochemical changes that are common in patients harboring recessive RYR1 and SELENON variants, including depletion of transcripts encoding proteins involved in skeletal muscle calcium homeostasis, increased levels of Class II histone deacetylases (HDACs) and DNA methyltransferases. CpG methylation analysis of genomic DNA of patients with RYR1 and SELENON variants identified >3,500 common aberrantly methylated genes, many of which are involved in calcium signaling. These results provide the proof of concept for the potential use of drugs targeting HDACs and DNA methyltransferases to treat patients with specific forms of congenital myopathies.

First-Line Therapy for Metastatic Soft Tissue Sarcoma.

OPINION STATEMENT: Soft tissue sarcomas are rare cancers with an expected incidence of about 14,000 new cases in 2018, and account for less than 1% of all cancers. It includes in excess of 75 heterogeneous subtypes with varying biology, molecular aberrations, and variable response to treatment. Because of the rarity of these tumors and the many different subtypes, there is no large-scale data to guide treatment, and hence the need for a multidisciplinary individualized approach to treatment, preferably at a high-volume tertiary referral center. For localized disease, surgery with or without radiation is the preferred treatment. In metastatic disease, the longest track record is with use of anthracyclines, either alone or in combination with ifosfamide, but the median overall survival even with combination was just over a year. There have been recent advances in understanding the heterogeneity of these tumors and the need for an individualized approach. With that new knowledge, recent approvals of trabectedin, eribulin, and pazopanib have been limited to some select histologic subtypes with improved outcomes. More recently, immunotherapy has been tested in select histotypes of sarcoma with encouraging activity and has led to further evaluation in combination with immunotherapeutic agents, as well as with chemotherapy and radiation treatments. Here, in this article, we summarize the data of the currently approved therapies in metastatic soft tissue sarcoma, with the principal focus on first-line therapies. We also review the recent encouraging data with PDGFR-targeted antibody (olaratumab) with doxorubicin which showed an impressive improvement in overall survival in phase II study. Molecular characterization of sarcoma subtypes will likely improve understanding of these very diverse tumors and improve target characterization. The ongoing efforts in better understanding these rare tumors hold the key to make a difference in the outcome of these patients.

The cholesterol metabolite 27-hydroxycholesterol stimulates cell proliferation via ERß in prostate cancer cells.

BACKGROUND: For every six men, one will be diagnosed with prostate cancer (PCa) in their lifetime. Estrogen receptors (ERs) are known to play a role in prostate carcinogenesis. However, it is unclear whether the estrogenic effects are mediated by estrogen receptor α (ERα) or estrogen receptor ß (ERß). Although it is speculated that ERα is associated with harmful effects on PCa, the role of ERß in PCa is still ill-defined. The cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC) has been found to bind to ERs and act as a selective ER modulator (SERM). Increased 27-OHC levels are found in individuals with hypercholesterolemia, a condition that is suggested to be a risk factor for PCa. METHODS: In the present study, we determined the extent to which 27-OHC causes deleterious effects in the non-tumorigenic RWPE-1, the low tumorigenic LNCaP, and the highly tumorigenic PC3 prostate cancer cells. We conducted cell metabolic activity and proliferation assays using MTS and CyQUANT dyes, protein expression analyses via immunoblots and gene expression analyses via RT-PCR. Additionally, immunocytochemistry and invasion assays were performed to analyze intracellular protein distribution and quantify transepithelial cell motility. RESULTS: We found that incubation of LNCaP and PC3 cells with 27-OHC significantly increased cell proliferation. We also demonstrate that the ER inhibitor ICI 182,780 (fulvestrant) significantly reduced 27-OH-induced cell proliferation, indicating the involvement of ERs in proliferation. Interestingly, ERß levels, and to a lesser extent ERα, were significantly increased following incubation of PCa cells with 27-OHC. Furthermore, in the presence of the ERß specific inhibitor, PHTPP, 27-OHC-induced proliferation is attenuated. CONCLUSIONS: Altogether, our results show for the first time that 27-OHC, through ER activation, triggers deleterious effect in prostate cancer cell lines. We propose that dysregulated levels of 27-OHC may trigger or exacerbate prostate cancer via acting on ERß.

Ozone-derived Oxysterols Affect Liver X Receptor (LXR) Signaling: A POTENTIAL ROLE FOR LIPID-PROTEIN ADDUCTS.

When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -ß and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3 Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects.

Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study.

UNLABELLED: Enhancing antiviral host defense responses through nutritional supplementation would be an attractive strategy in the fight against influenza. Using inoculation with live attenuated influenza virus (LAIV) as an infection model, we have recently shown that ingestion of sulforaphane-containing broccoli sprout homogenates (BSH) reduces markers of viral load in the nose. To investigate the systemic effects of short-term BSH supplementation in the context of LAIV-inoculation, we examined peripheral blood immune cell populations in non-smoking subjects from this study, with a particular focus on NK cells. We carried out a randomized, double-blinded, placebo-controlled study measuring the effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on peripheral blood mononuclear cell responses to a standard nasal vaccine dose of LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2, day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated, and assessed for surface markers, intracellular markers, and cytotoxic potential by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while significantly increasing CD16 expression and cytotoxic potential (on day2). BSH supplementation further increased LAIV-induced granzyme B production (day2) in NK cells compared to ASH and in the BSH group granzyme B levels appeared to be negatively associated with influenza RNA levels in nasal lavage fluid cells. We conclude that nasal influenza infection may induce complex changes in peripheral blood NK cell activation, and that BSH increases virus-induced peripheral blood NK cell granzyme B production, an effect that may be important for enhanced antiviral defense responses. TRIAL REGISTRATION: ClinicalTrials.gov NCT01269723.

27-Hydroxycholesterol stimulates cell proliferation and resistance to docetaxel-induced apoptosis in prostate epithelial cells.

Although the causes of prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are not known, the role of oxidative stress, aging, and diet are suspected to increase the incidence of prostate complications. The cholesterol oxidation derivative (oxysterol) 27-hydroxycholesterol (27-OHC) is the most prevalent cholesterol metabolite in the blood. As aging, oxidative stress, and hypercholesterolemia are associated with increased risk of PCa and BPH, and because 27-OHC levels are also increased with aging, hypercholesterolemia, and oxidative stress, determining the role of 27-OHC in the progression of PCas and BPH is warranted. In this study, we determined the effect of 27-OHC in human prostate epithelial cells RWPE-1. We found that 27-OHC stimulates proliferation and increases androgen receptor (AR) transcriptional activity. 27-OHC also increased prostate-specific antigen expression and enhanced AR binding to the androgen response element compared to controls. Silencing AR expression with siRNA markedly reduced the 27-OHC-induced proliferation. Furthermore, 27-OHC blocked docetaxel-induced apoptosis. Altogether, our results suggest that 27-OHC may play an important role in PCa and BPH progression by promoting proliferation and suppressing apoptosis.

Respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants.

The respiratory epithelium functions as a central orchestrator to initiate and organize responses to inhaled stimuli. Proteases and antiproteases are secreted from the respiratory epithelium and are involved in respiratory homeostasis. Modifications to the protease/antiprotease balance can lead to the development of lung diseases such as emphysema or chronic obstructive pulmonary disease. Furthermore, altered protease/antiprotease balance, in favor for increased protease activity, is associated with increased susceptibility to respiratory viral infections such as influenza virus. However, nutritional antioxidants induce antiprotease expression/secretion and decrease protease expression/activity, to protect against viral infection. As such, this review will elucidate the impact of this balance in the context of respiratory viral infection and lung disease, to further highlight the role epithelial cell-derived proteases and antiproteases contribute to respiratory immune function. Furthermore, this review will offer the use of nutritional antioxidants as possible therapeutics to boost respiratory mucosal responses and/or protect against infection.

Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood.

Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56(+)CD45(+)CD3(-)CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine-fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (<20%) or high (>50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting a bias in the SK pathway towards proliferation and migration, activities supported by S1P.

Effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study.

BACKGROUND: Smokers have increased susceptibility and altered innate host defense responses to influenza virus infection. Broccoli sprouts are a source of the Nrf2 activating agentsulforaphane, and short term ingestion of broccoli sprout homogenates (BSH) has been shown to reduce nasal inflammatory responses to oxidant pollutants. OBJECTIVES: Assess the effects of BSH on nasal cytokines, virus replication, and Nrf2-dependent enzyme expression in smokers and nonsmokers. METHODS: We conducted a randomized, double-blind, placebo-controlled trial comparing the effects of BSH on serially sampled nasal lavage fluid (NLF) cytokines, viral sequence quantity, and Nrf2-dependent enzyme expression in NLF cells and biopsied epithelium. Healthy young adult smokers and nonsmokers ingested BSH or placebo (alfalfa sprout homogenate) for 4 days, designated Days -1, 0, 1, 2. On Day 0 they received standard vaccine dose of live attenuated influenza virus (LAIV) intranasally. Nasal lavage fluids and nasal biopsies were collected serially to assess response to LAIV. RESULTS: In area under curve analyses, post-LAIV IL-6 responses (P = 0.03) and influenza sequences (P = 0.01) were significantly reduced in NLF from BSH-treated smokers, while NAD(P)H: quinoneoxidoreductasein NLF cells was significantly increased. In nonsmokers, a similar trend for reduction in virus quantity with BSH did not reach statistical significance. CONCLUSIONS: In smokers, short term ingestion of broccoli sprout homogenates appears to significantly reduce some virus-induced markers of inflammation, as well as reducing virus quantity. Nutritional antioxidant interventions have promise as a safe, low-cost strategy for reducing influenza risk among smokers and other at risk populations. TRIAL REGISTRATION: ClinicalTrials.gov NCT01269723.

Regulation and activity of secretory leukoprotease inhibitor (SLPI) is altered in smokers.

A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection.

Sulforaphane induces SLPI secretion in the nasal mucosa.

Cells lining the respiratory tract are equipped with mechanisms that dampen the effects of oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a mediator involved in regulating oxidative stress. Recent data indicate Nrf2 also controls expression of secretory leukocyte protease inhibitor (SLPI). Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, enhances Nrf2 activity. Therefore, we hypothesized that SFN supplementation induces SLPI secretion in the nasal mucosa in an Nrf2 dependent manner. Healthy nonsmoking adults ingested SFN-containing broccoli shake homogenate (BSH) for 3 consecutive days. Nasal lavage fluid (NLF) was collected before and after BSH ingestion and analyzed for SLPI protein levels. In follow up in vitro experiments, differentiated primary nasal epithelial cells were used to evaluate the relationship between SFN, Nrf2, and SLPI. Epithelial cells were transduced with Nrf2-specific shRNA to examine the regulatory role of Nrf2 on SLPI expression. Supplementation with BSH significantly increased SLPI levels in NLF. SFN supplementation in vitro significantly enhanced SLPI secretion and these effects were significantly decreased in cells transduced with Nrf2-specific shRNA. Our data support a relationship between nutritional supplementation, Nrf2 activation, and SLPI secretion. Therefore, ingestion of SFN-containing foods has therapeutic potential to augment SLPI expression in the nasal mucosa.

Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection.

Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA) is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI). Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs) to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

Diesel exhaust exposure and nasal response to attenuated influenza in normal and allergic volunteers.

RATIONALE: Diesel exhaust enhances allergic inflammation, and pollutants are associated with heightened susceptibility to viral respiratory infections. The effects of combined diesel and virus exposure in humans are unknown. OBJECTIVES: Test whether acute exposure to diesel modifies inflammatory responses to influenza virus in normal humans and those with allergies. METHODS: We conducted a double-blind, randomized, placebo-controlled study of nasal responses to live attenuated influenza virus in normal volunteers and those with allergic rhinitis exposed to diesel (100 µg/m(3)) or clean air for 2 hours, followed by standard dose of virus and serial nasal lavages. Endpoints were inflammatory mediators (ELISA) and virus quantity (quantitative reverse-transcriptase polymerase chain reaction). To test for exposure effect, we used multiple regression with exposure group (diesel vs. air) as the main explanatory variable and allergic status as an additional factor. MEASUREMENTS AND MAIN RESULTS: Baseline levels of mediators did not differ among groups. For most postvirus nasal cytokine responses, there was no significant diesel effect, and no significant interaction with allergy. However, diesel was associated with significantly increased IFN-γ responses (P = 0.02), with no interaction with allergy in the regression model. Eotaxin-1 (P = 0.01), eosinophil cationic protein (P < 0.01), and influenza RNA sequences in nasal cells (P = 0.03) were significantly increased with diesel exposure, linked to allergy. CONCLUSIONS: Short-term exposure to diesel exhaust leads to increased eosinophil activation and increased virus quantity after virus inoculation in those with allergic rhinitis. This is consistent with previous literature suggesting a diesel "adjuvant" effect promoting allergic inflammation, and our data further suggest this change may be associated with reduced virus clearance.Clinical trial registered with www.clinicaltrials.gov (NCT00617110).

A conserved glutamate controls the commitment to acyl-adenylate formation in asparagine synthetase.

Inhibitor docking studies have implicated a conserved glutamate residue (Glu-348) as a general base in the synthetase active site of the enzyme asparagine synthetase B from Escherichia coli (AS-B). We now report steady-state kinetic, isotope transfer, and positional isotope exchange experiments for a series of site-directed AS-B mutants in which Glu-348 is substituted by conservative amino acid replacements. We find that formation of the ß-aspartyl-AMP intermediate, and therefore the eventual production of asparagine, is dependent on the presence of a carboxylate side chain at this position in the synthetase active site. In addition, Glu-348 may also play a role in mediating the conformational changes needed to (i) coordinate, albeit weakly, the glutaminase and synthetase activities of the enzyme and (ii) establish the structural integrity of the intramolecular tunnel along which ammonia is translocated. The importance of Glu-348 in mediating acyl-adenylate formation contrasts with the functional role of the cognate residues in ß-lactam synthetase (BLS) and carbapenem synthetase (CPS) (Tyr-348 and Tyr-345, respectively), which both likely evolved from asparagine synthetase. Given the similarity of the chemistry catalyzed by AS-B, BLS, and CPS, our work highlights the difficulty of predicting the functional outcome of single site mutations on enzymes that catalyze almost identical chemical transformations.

A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase.

The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of L-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure-function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability.

Measuring sense of community: a view from the streets.

Increasing social capital in poor communities has become a common pursuit among policy-makers, social scientists and community practitioners alike. In this paper, we examine one dimension of social capital, sense of community (SOC), and argue that the dominant instruments used by scholars to measure it are limited. We examine qualitative data from surveys, focus groups and interviews with community leaders and residents in Baltimore, Maryland, to understand how community members conceptualize SOC and how they might measure it. We conclude with a discussion about how our findings can inform and strengthen future community-based research and community building initiatives.