International Society for Extracellular Vesicles Workshop. QuantitatEVs: multiscale analyses, from bulk to single extracellular vesicle.

The 'QuantitatEVs: multiscale analyses, from bulk to single vesicle' workshop aimed to discuss quantitative strategies and harmonized wet and computational approaches toward the comprehensive analysis of extracellular vesicles (EVs) from bulk to single vesicle analyses with a special focus on emerging technologies. The workshop covered the key issues in the quantitative analysis of different EV-associated molecular components and EV biophysical features, which are considered the core of EV-associated biomarker discovery and validation for their clinical translation. The in-person-only workshop was held in Trento, Italy, from January 31st to February 2nd, 2023, and continued in Milan on February 3rd with "Next Generation EVs", a satellite event dedicated to early career researchers (ECR). This report summarizes the main topics and outcomes of the workshop.

miR-210-3p enriched extracellular vesicles from hypoxic neuroblastoma cells stimulate migration and invasion of target cells.

BACKGROUND: Tumor hypoxia stimulates release of extracellular vesicles (EVs) that facilitate short- and long-range intercellular communication and metastatization. Albeit hypoxia and EVs release are known features of Neuroblastoma (NB), a metastasis-prone childhood malignancy of the sympathetic nervous system, whether hypoxic EVs can facilitate NB dissemination is unclear. METHODS: Here we isolated and characterized EVs from normoxic and hypoxic NB cell culture supernatants and performed microRNA (miRNA) cargo analysis to identify key mediators of EVs biological effects. We then validated if EVs promote pro-metastatic features both in vitro and in an in vivo zebrafish model. RESULTS: EVs from NB cells cultured at different oxygen tensions did not differ for type and abundance of surface markers nor for biophysical properties. However, EVs derived from hypoxic NB cells (hEVs) were more potent than their normoxic counterpart in inducing NB cells migration and colony formation. miR-210-3p was the most abundant miRNA in the cargo of hEVs; mechanistically, overexpression of miR-210-3p in normoxic EVs conferred them pro-metastatic features, whereas miR-210-3p silencing suppressed the metastatic ability of hypoxic EVs both in vitro and in vivo. CONCLUSION: Our data identify a role for hypoxic EVs and their miR-210-3p cargo enrichment in the cellular and microenvironmental changes favoring NB dissemination.

Clinical and immunological evaluation of 12-month azithromycin therapy in chronic lung allograft rejection.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the leading cause of morbidity/mortality in lung-transplant recipients (LTRs). Recent studies demonstrated that azithromycin (AZI) can improve graft function in BOS. We here investigated whether a 12-month course of AZI could more efficiently impact the course of BOS if administered early in BOS development. METHODS: Using a retrospective study, we examined AZI effects on graft function in 62 LTRs: 25 with potential BOS (BOS 0-p) and 37 with BOS grade 1-3. Response was defined as a ≥ 10% FEV(1) increase. Bronchoalveolar (BAL) neutrophilia and levels of IL-8, 8-isoprostane and other plasma cytokines were analyzed as parameters of lung or systemic inflammation. RESULTS: After 12-month AZI, 13 patients were responders, 35 had graft function stabilization, and 14 further deteriorated. The frequency of responders was significantly higher in LTRs with BOS 0-p (44%) than in those with BOS grade 1-3 (6%). No association was found between BAL features and AZI response while a significant decrease in plasma levels of IL-8, MCP-1, I-309, MIP-1α, and TNF-α was detected. CONCLUSIONS: Long-term AZI can improve or stabilize lung graft function in LTRs with BOS, but the treatment impacts the course of the disease more efficiently if administered in BOS 0-p.

Foxp3 expressing CD4+ CD25+ and CD8+CD28- T regulatory cells in the peripheral blood of patients with lung cancer and pleural mesothelioma.

The role of T regulatory (Treg) cells in human cancer has not yet been clarified. We assessed the presence and function of CD4+ and CD8+ Treg cell subsets in the peripheral blood of patients with lung cancer (LC) and pleural mesothelioma (PM). We found a low but significant increase in the number of CD4+ T cells with phenotype and functional features of Treg cells in LC patients compared to normal healthy controls (NHC). Furthermore, total CD4+ T cells from LC patients proliferated less than cells from controls, suggesting that the increase in the CD4+ Treg cell pool has functional importance. LC patients also showed an expansion of the CD8+CD28- T cell subset and these cells expressed Foxp3 mRNA, as recently observed in alloantigen-specific CD8+CD28- T suppressor cells. No variation of peripheral Treg cell subsets was found in patients with PM, a disease with a predominantly localized nature. However, the lack of correlation between cancer stage and the number or the function of peripheral Treg cells in LC patients refuted the hypothesis that these cells are involved in tumor spreading. A possible involvement of the peripheral Treg cell pool in cancer development and/or in inducing systemic immunosuppression in LC patients can be hypothesized.

Interleukin-8 production induced by the endozepine triakontatetraneuropeptide in human neutrophils: role of calcium and pharmacological investigation of signal transduction pathways.

The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca(2+)](i)) changes and is chemotactic for human neutrophils (PMNs). Because interleukin-8 (IL-8) production is Ca(2+) dependent and can be induced by chemotactic stimuli, we have investigated the ability of TTN to induce IL-8 production in PMNs, as well as the signal transduction mechanisms involved. Our results show that TTN increases IL-8 release and IL-8 mRNA expression in a concentration- and time-dependent fashion, and these effects are prevented by the Ca(2+) chelator BAPTA-AM. TTN-induced [Ca(2+)](i) changes and IL-8 mRNA expression are sensitive to pertussis toxin, to the phospholipase C (PLC) inhibitor U73122 (but not to its inactive analogue U73343) and to the protein kinase C (PKC) inhibitor calphostin C. It is therefore suggested that TTN-induced IL-8 production in human PMNs results from a G protein-operated, PLC-activated [Ca(2+)](i) rise, and PKC contributes to this effect. These findings further support the possible role of TTN in the modulation of the inflammatory processes.

Regulatory CD4+CD25+ T cells in the peripheral blood of lung transplant recipients: correlation with transplant outcome.

BACKGROUND: The subset of CD4+CD25+ regulatory T cells, recently identified in humans, may play a central role in the regulation of immune tolerance to graft survival. METHODS: This study assesses the frequency and functional profile of CD4+CD25+CD69- cells in the peripheral blood of lung transplant recipients (>3 years from transplantation), 10 of whom were in a stable clinical condition and 11 of whom demonstrated chronic rejection (bronchiolitis obliterans syndrome). We also studied a group of seven healthy subjects. RESULTS: The frequency of CD4+ T cells expressing CD25 (CD4+CD25+) and the highest levels (CD25) were lower in patients with bronchiolitis obliterans syndrome compared with healthy subjects and subjects in a stable clinical condition (P < or = 0.01). Purified CD4+CD25+ cells exhibited a regulatory functional profile in vitro: they were hyporesponsive, suppressed the proliferation of CD4+CD25- cells, and produced interleukin-10. CONCLUSION: These results provide in vivo evidence that peripheral CD4+CD25+ T cells may represent an important regulatory subset in lung transplantation.

Bronchoalveolar lavage cytokine profile in a cohort of lung transplant recipients: a predictive role of interleukin-12 with respect to onset of bronchiolitis obliterans syndrome.

BACKGROUND: Bronchiolitis obliterans syndrome is the main long-term complication of lung transplantation that limits survival of lung transplant patients. Its pathophysiologic mechanisms are still poorly understood but it seems to result from a chronic immunologic/inflammatory insult leading to excessive fibroproliferation. The aim of this longitudinal study of 44 lung recipients was to determine whether a number of bronchoalveolar lavage and clinical variables are associated with a higher risk of developing bronchiolitis obliterans syndrome. METHODS: Bronchoalveolar lavage studies involved assessment of several cytokines including: interleukin-8, monocyte chemoattractant protein-1, regulated-upon-activation normal T cell expressed and secreted (RANTES), gamma-interferon, interleukin-12, interleukin-10 and transforming growth factor-beta. RESULTS: The predictivity of bronchoalveolar lavage (BAL) features with respect to onset of bronchiolitis obliterans syndrome was assessed by the Cox regression model. Among clinical variables, bacterial and viral infections were found to significantly predict occurrence of bronchiolitis obliterans syndrome (hazard ratio [HR] for bacterial infection: 13.044, 95% confidence interval [CI] 1.34 to 126.69, p = 0.027; HR for viral infections: 4.88, 95% CI 1.004 to 22.87, p = 0.05). Among BAL variables, only IL-12 was significantly predictive of bronchiolitis obliterans syndrome (HR 0.956, 95% CI 0.901 to 1.01, p = 0.03). In addition, in a sub-group cross-sectional analysis, bronchiolitis obliterans syndrome patients were compared with clinically stable patients, and significant increases in median levels of interleukin-8 and monocyte chemoattractant protein-1 BAL fluid were detected. CONCLUSIONS: These findings support the contention that interleukin-12 plays a role in the modulation of the local pro-/anti-fibrotic balance of allograft airways.

Comparison of a whole-blood interferon-gamma assay and tuberculin skin testing in patients with active tuberculosis and individuals at high or low risk of Mycobacterium tuberculosis infection.

BACKGROUND: QuantiFeron-TB (QIFN) is a whole-blood interferon-;gamma assay for the recognition of cell-mediated immune response to Mycobacterium tuberculosis infection. OBJECTIVES: To compare the QIFN assay with the tuberculin skin test (TST) in patients with newly diagnosed culture-proven tuberculosis (TB) and healthy volunteers with high or low risk of latent M tuberculosis infection and to identify factors associated with discordance between tests. METHOD: Two-hundred fifty-eight subjects underwent both assays. All participants completed a detailed questionnaire, and data from TB patients' medical records were collected. RESULTS: In the entire study population, agreement between tests was moderate and the correlation between the magnitude of QIFN response and the TST induration diameter was significant. In volunteers with no known risk of exposure to M tuberculosis, the specificity of the assays was comparable. However, in subjects with active TB or those vaccinated with bacille Calmette-Guérin, the QIFN assay detected more reactors than did the TST. In these individuals, agreement between assays was poor and no correlation or only a weak correlation was found between the diameter of TST induration and the magnitude of the interferon-gamma responses. CONCLUSIONS: The sensitivity of the QIFN assay is greater than that of the TST in patients with active TB before the initiation of anti-TB chemotherapy, but its specificity is influenced more by bacille Calmette-Guérin vaccination. The QIFN assay may provide an improvement over the current practice of the use of the TST to support diagnosis of active M tuberculosis infection in the clinic; however, QIFN cannot be considered an adequate replacement for the TST in the screening for latent infection.

Intracellular calcium changes induced by the endozepine triakontatetraneuropeptide in human polymorphonuclear leukocytes: role of protein kinase C and effect of calcium channel blockers.

BACKGROUND: The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca++]i) changes followed by activation in human polymorphonuclear leukocytes (PMNs). The present study was undertaken to investigate the role of protein kinase (PK) C in the modulation of the response to TTN by human PMNs, and to examine the pharmacology of TTN-induced Ca++ entry through the plasma membrane of these cells. RESULTS: The PKC activator 12-O-tetradecanoylphorbol-13-acetate (PMA) concentration-dependently inhibited TTN-induced [Ca++]i rise, and this effect was reverted by the PKC inhibitors rottlerin (partially) and Ro 32-0432 (completely). PMA also inhibited TTN-induced IL-8 mRNA expression. In the absence of PMA, however, rottlerin (but not Ro 32-0432) per se partially inhibited TTN-induced [Ca++]i rise. The response of [Ca++]i to TTN was also sensitive to mibefradil and flunarizine (T-type Ca++-channel blockers), but not to nifedipine, verapamil (L-type) or omega-conotoxin GVIA (N-type). In agreement with this observation, PCR analysis showed the expression in human PMNs of the mRNA for all the alpha1 subunits of T-type Ca++ channels (namely, alpha1G, alpha1H, and alpha1I). CONCLUSIONS: In human PMNs TTN activates PKC-modulated pathways leading to Ca++ entry possibly through T-type Ca++ channels.

Cytokine profile of broncho-alveolar lavage in BOOP and UIP.

BACKGROUND: The aim of our study was to compare clinical and BAL features of patients with bronchiolitis obliterans organizing pneumonia (BOOP) with those of patients with usual interstitial pneumonia (UIP) and control subjects. PATIENTS AND METHODS: This study reports on 14 patients with idiopathic BOOP. Diagnosis was made upon histology. Lung function tests were mostly normal. Chest X-ray and CT showed always a patchy consolidation, often associated with ground glass pattern. BAL was performed for cytology and for ELISA assessment of several cytokines (IL8, ILI0, IL12, gamma-interferon, IL 18, monocyte chemoattractant protein- 1). RESULTS: Cytology of BAL in BOOP showed a pattern of lymphocytic alveolitis (Lymphocytes: 0.36 x 10(6)/ml) associated with an increase in neutrophil and eosinophil counts (0.13 and 0.04 x 10(6)/ml respectively). Mean BALf levels in pg/ml of MCP-1, IL12 and IL18 were significantly increased in BOOP with respect to controls and UIP patients, while in UIP patients only a significant increase of IL8, MCP-1 and IL18 with respect to controls was detected. In addition, BALf levels of IL10, an anti-inflammatory cytokine, were significantly higher in BOOP patients with respect to controls and UIP patients. CONCLUSION: These findings are consistent with a marked degree of macrophage and lymphocyte activation in BOOP with an expansion of T helper-1 response. The concomitant increase of IL10 could be related to a limitation of the inflammatory process and the fibrotic evolution typical of this clinical picture.

BAL cytokine profile in different interstitial lung diseases: a focus on systemic sclerosis.

BACKGROUND AND AIM: Fibrosing alveolitis develops in up to 80% of systemic sclerosis patients (SSc) but progression to end stage fibrosis occurs in about 15% of cases. Mechanisms leading to the process remain mostly unknown. We compared cytokine profiles of broncho-alveolar lavage fluids (BAL-f) from patients with SSc associated interstitial lung disease (SSc-ILD) (n. 34), idiopathic pulmonary fibrosis (IPF) (n. 13), stage II sarcoidosis (n. 14) and 9 controls. METHODS: Interleukin (IL) 8, monocyte chemoattractant protein 1 (MCP-1), gamma-interferon (IFN-gamma), IL12, IL18 and IL10 and transforming growth factor-beta (TGF-beta) were assessed by ELISA in concentrated BAL-f. RESULTS: Levels of IL8 and MCP-1 were significantly elevated in SSc-ILD and in IPF as compared with controls (Mann Whitney test p < 0.05), while MCP-1 values were significantly lower in SSc-ILD than in IPF. A significant correlation between neutrophils and IL8 levels (p = 0.047), as well as between eosinophils and MCP-1 levels (p = 0.004) was also observed. IFN-gamma levels were slightly higher than normal only in sarcoidosis (p = 0.06), whereas IL12 levels increased both in sarcoidosis and SSc-ILD (p < 0.05). No differences were found in IL18 and TGF-beta levels. Finally, IL10 levels were higher in SSc-ILD and sarcoidosis than in controls and IPF (p < 0.05). CONCLUSION: BAL-f cytokine profile differentiates ILD associated with SSc from IPF. The lower expression of MCP-1 and the higher expression of the anti-fibrotic IL12 and the anti-inflammatory IL10, observed both in sarcoidosis and in SSc-ILD, could account for the better prognosis of these ILDs. Further longitudinal studies are required to confirm whether a different cytokine phenotype may be considered predictive of clinical outcome in SSc-ILD.

New developments in antibacterial choice for lower respiratory tract infections in elderly patients.

Elderly patients are at increased risk of developing lower respiratory tract infections compared with younger patients. In this population, pneumonia is a serious illness with high rates of hospitalisation and mortality, especially in patients requiring admission to intensive care units (ICUs). A wide range of pathogens may be involved depending on different settings of acquisition and patient's health status. Streptococcus pneumoniae is the most common bacterial isolate in community-acquired pneumonia, followed by Haemophilus influenzae, Moraxella catarrhalis and atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae. However, elderly patients with comorbid illness, who have been recently hospitalised or are residing in a nursing home, may develop severe pneumonia caused by multidrug resistant staphylococci or pneumococci, and enteric Gram-negative bacilli, including Pseudomonas aeruginosa. Moreover, anaerobes may be involved in aspiration pneumonia. Timely and appropriate empiric treatment is required in order to enhance the likelihood of a good clinical outcome, prevent the spread of antibacterial resistance and reduce the economic impact of pneumonia. International guidelines recommend that elderly outpatients and inpatients (not in ICU) should be treated for the most common bacterial pathogens and the possibility of atypical pathogens. The algorithm for therapy is to use either a selected beta-lactam combined with a macrolide (azithromycin or clarithromycin), or to use monotherapy with a new anti-pneumococcal quinolone, such as levofloxacin, gatifloxacin or moxifloxacin. Oral (amoxicillin, amoxicillin/clavulanic acid, cefuroxime axetil) and intravenous (sulbactam/ampicillin, ceftriaxone, cefotaxime) beta-lactams are agents of choice in outpatients and inpatients, respectively. For patients with severe pneumonia or aspiration pneumonia, the specific algorithm is to use either a macrolide or a quinolone in combination with other agents; the nature and the number of which depends on the presence of risk factors for specific pathogens. Despite these recommendations, clinical resolution of pneumonia in the elderly is often delayed with respect to younger patients, suggesting that optimisation of antibacterial therapy is needed. Recently, some new classes of antibacterials, such as ketolides, oxazolidinones and streptogramins, have been developed for the treatment of multidrug resistant Gram-positive infections. However, the efficacy and safety of these agents in the elderly is yet to be clarified. Treatment guidelines should be modified on the basis of local bacteriology and resistance patterns, while dosage and/or administration route of each antibacterial should be optimised on the basis of new insights on pharmacokinetic/pharmacodynamic parameters and drug interactions. These strategies should be able to reduce the occurrence of risk factors for a poor clinical outcome, hospitalisation and death.

Systemic inflammatory response and downmodulation of peripheral CD25+Foxp3+ T-regulatory cells in patients undergoing radiofrequency thermal ablation for lung cancer.

Radiofrequency thermal ablation (RFTA) is a local tumor-destructing technique that can potentially modulate the host immune response through mechanisms that are not clearly defined. We assessed whether RFTA could affect multiple systemic inflammatory and immunological parameters, including CD25+Foxp+ cells, in patients with primary or metastatic lung tumors. Three days after RFTA, a moderate and temporary systemic inflammatory response developed, as demonstrated by the increase in peripheral neutrophils and monocytes and in plasma levels of proinflammatory chemokines (MIP-1alpha, MIP-1beta, eotaxin, and interleukin[IL]-8) and acute phase reactants (complement C3 and C4, serum amyloid, alpha1 antichymotrypsin, and C-reactive protein). Moreover, we found a concomitant release of the anti-inflammatory factor IL-10. Thirty days after RFTA, a significant reduction in CD25+Foxp3+ counts with an increase in CD4+ T-cell proliferation and number of interferon-gamma-secreting cells was observed. The reduction in CD25+Foxp3+ cells lasted up to 90 days after treatment. The use of RFTA in lung cancer patients has an immunomodulatory activity: it induces a self-limiting systemic inflammation early and later a reduction of circulating CD25+Foxp3+ Tregs. In addition to tumor ablation, downmodulation of this regulatory subset might be an important mechanism involved in the long-term clinical efficacy of RFTA.

Indoleamine 2,3-dioxygenase in lung allograft tolerance.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the degradation of tryptophan (Try) to kynurenine (Kyn), is thought to suppress T-cell activity. Although a few experimental studies have suggested a role for IDO in graft acceptance, human data are scarce and inconclusive. We sought to establish whether, in lung transplant recipients (LTRs), plasma IDO activity mirrors the level of graft acceptance. METHODS: We measured the plasma Kyn/Try ratio, reflecting IDO activity, by high-performance liquid chromatography (HPLC) in 90 LTRs, including 26 patients who were still functionally/clinically stable for >36 post-transplant months (stable LTRs) and 64 LTRs with bronchiolitis obliterans syndrome (BOS, Grades 0-p to 3). Twenty-four normal healthy controls (NHCs) were also included. RESULTS: The Kyn/Try ratio in stable LTRs resembled that observed in NHCs, whereas, unexpectedly, patients with BOS, who had lower counts of peripheral CD4(+) T-regulatory cells and tolerogenic plasmacytoid dendritic cells than stable LTRs, showed an increased plasma Kyn/Try ratio compared with both NHCs and stable LTRs. IDO expression by in vitro-stimulated peripheral blood mononuclear cells (PBMC) did not vary between BOS and stable LTRs. Furthermore, BOS patients displayed signs of chronic systemic inflammation (increased plasma levels of interleukin-8 and tumor necrosis factor-alpha) and higher T-cell activation (increased frequency of peripheral interferon-gamma-producing clones). CONCLUSIONS: Our results suggest that, in vivo, in lung transplantation, plasma IDO activity does not reflect the degree of lung graft acceptance, but instead is correlated with the degree of chronic inflammation.

2-DE and LC-MS/MS for a comparative proteomic analysis of BALf from subjects with different subsets of inflammatory myopathies.

The protein profiles of bronchoalveolar lavage fluid (BALf) of patients belonging to three selected subsets of Polymyositis/Dermatomyositis (PM/DM) have been compared by using a combination of 2-DE and MALDI-TOF/MS or LC-MS/MS. Our study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype. From among the 323+/-51 protein spots that may represent the most highly expressed proteins in BALf of these patients, 24 unique spots were isolated and proteins identified. In particular, 9 spots were present in BALf of PM/DM patients only; 12 spots were exclusive of Overlap patients and 3 spots of AS patients. From among the proteins identified, a few were classified as cytoskeletal proteins, others were involved in oxidative stress and a number of proteins were associated with general metabolic activity or immunological response and inflammation. This is the first study in which evidence is provided that a number of different proteins are expressed in different subsets of PM/DM and supports our contention that the proteomic approach would be beneficial in discovering molecules which could represent possible prognostic factors of these rare pathologies.

Bronchoalveolar lavage fluid proteome in bronchiolitis obliterans syndrome: possible role for surfactant protein A in disease onset.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) affects long-term survival of lung transplant (Tx) recipients (LTRs), with no consistently effective treatment strategy. Identifying early markers of BOS is of paramount importance for improving graft survival. METHODS: We used 2-dimensional gel electrophoresis and protein identification by mass spectrometry to compare the protein profile of bronchoalveolar lavage fluid (BALf) in two groups of LTRs: one composed of patients with BOS and the other composed of patients with good graft function at >5 years post-surgery (stable LTRs). Based on the hypothesis that only proteins of lung origin could represent reliable BOS markers, we also evaluated paired plasma samples. Proteins of interest were also assessed in the BALf of control subjects and results confirmed by dot- blot analysis. RESULTS: Among 11 differentially expressed proteins, we identified 2 locally produced factors: peroxiredoxin II (PRXII), exclusively expressed in BOS; and surfactant protein A (SP-A), expressed consistently less in BOS patients than in stable LTRs. PRXII expression was never observed in BALf from control subjects, whereas SP-A was present in higher amounts compared with stable LTRs and BOS patients. Finally, the time course of SP-A was studied in 5 LTRs who subsequently developed BOS. A reduction in BALf SP-A content was detectable early after Tx, preceding BOS onset in 4 of 5 patients. CONCLUSIONS: Our results suggest that testing SP-A levels in BALf could predict LTR patients who are at higher risk of BOS development.

Human CD4+CD25+ regulatory T cells selectively express tyrosine hydroxylase and contain endogenous catecholamines subserving an autocrine/paracrine inhibitory functional loop.

CD4+CD25+ regulatory T lymphocytes (Tregs) are specialized T cells playing a key role in the control of immune homeostasis. Here, we show that human Tregs constitutively express tyrosine hydroxylase (TH, EC 1.14.16.2), the rate-limiting enzyme in the synthesis of catecholamines, and contain substantial amounts of dopamine, norepinephrine, and epinephrine, which are released upon treatment with reserpine. Catecholamine release results in reduced production of interleukin-10 and transforming growth factor-beta by Tregs, and in down-regulation of Treg-dependent inhibition of effector T-lymphocyte (Teff) proliferation, which occurs without affecting the production of tumor necrosis factor-alpha or interferon-gamma. Tregs and Teffs express on the cell membrane both D1-like and D2-like dopaminergic receptors to a similar extent (12%-29% of the cells). Catecholamine-dependent down-regulation of Tregs is, however, selectively reversed by pharmacological blockade of dopaminergic D1-like receptors, which in Tregs only (and not in Teffs) are also expressed at the level of mRNA and are functionally coupled to intracellular production of cAMP. These findings indicate that in human Tregs endogenous catecholamines subserve an autocrine/paracrine loop involving dopaminergic pathways and resulting in down-regulation of Treg function.

Pharmacological analysis of signal transduction pathways required for mycobacterium tuberculosis-induced IL-8 and MCP-1 production in human peripheral monocytes.

Signalling cascades involved in chemokine production by human phagocytes following infection with Mycobacterium tuberculosis are still not defined. We used specific pharmacologic inhibitors to identify the signalling molecules which lead to interleukin (IL)-8 and MCP-1 production in human monocytes in response to M. tuberculosis infection. Inhibition of extracellular signal-regulated (ERK) or p38 mitogen-activated protein kinase by PD98059 and SB203580 respectively, significantly affected chemokine production. However, only the presence of both inhibitors completely blocked the release. A down-regulation of chemokine secretion was found in presence of inhibitors of protein kinase (PK)C and phospholipase C. Moreover, production depended on transcription activation via the nuclear factor-kappa B (NF-kappaB), as demonstrated by treatment with actinomycin D and caffeic acid phenethyl ester. In addition, activation of PKA and the phosphoinoside 3-kinase (PI-3k)/p70 ribosomal S6 kinase cascade was required to have maximal MCP-1 but not IL-8 production. In conclusion, this study provides evidence that multiple signal transduction pathways are involved in M. tuberculosis -induced chemokine secretion by human monocytes. Moreover, for the first time this report indicates that inhibitors of some signalling molecules are able to dissociate IL-8 from MCP-1 secretion. Differences in the regulatory pathways of chemokine production can potentially be exploited therapeutically.