A. Fietta

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 (CD25high) were lower in patients with bronchiolitis obliterans syndrome compared with healthy subjects and subjects in a stable clinical condition (P ≤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.

Analysis of bronchoalveolar lavage fluid proteome from systemic sclerosis patients with or without functional, clinical and radiological signs of lung fibrosis

Lung fibrosis is a major cause of mortality and morbidity in systemic sclerosis (SSc). However, its pathogenesis still needs to be elucidated. We examined whether the alteration of certain proteins in bronchoalveolar lavage fluid (BALF) might have a protective or a causative role in the lung fibrogenesis process. For this purpose we compared the BALF protein profile obtained from nine SSc patients with lung fibrosis (SScFib+) with that obtained from six SSc patients without pulmonary fibrosis (SScFib-) by two-dimensional gel electrophoresis (2-DE). Only spots and spot-trains that were consistently expressed in a different way in the two study groups were taken into consideration. In total, 47 spots and spot-trains, corresponding to 30 previously identified proteins in human BALF, showed no significant variation between SScFib+ patients and SScFib- patients, whereas 24 spots showed a reproducible significant variation in the two study groups. These latter spots corresponded to 11 proteins or protein fragments, including serum albumin fragments (13 spots), 5 previously recognized proteins (7 spots), and 4 proteins (3 spots) that had not been previously described in human BALF maps, namely calumenin, cytohesin-2, cystatin SN, and mitochondrial DNA topoisomerase 1 (mtDNA TOP1). Mass analysis did not determine one protein-spot. The two study groups revealed a significant difference in BALF protein composition. Whereas levels of glutathione S-transferase P (GSTP), Cu–Zn superoxide dismutase (SOD) and cystatin SN were downregulated in SScFib+ patients compared with SScFib- patients, we observed a significant upregulation of α1-acid glycoprotein, haptoglobin-α chain, calgranulin (Cal) B, cytohesin-2, calumenin, and mtDNA TOP1 in SScFib+ patients. Some of these proteins (GSTP, Cu–Zn SOD, and cystatin SN) seem to be involved in mechanisms that protect lungs against injury or inflammation, whereas others (Cal B, cytohesin-2, and calumenin) seem to be involved in mechanisms that drive lung fibrogenesis. Even if the 2-DE analysis of BALF did not provide an exhaustive identification of all BALF proteins, especially those of low molecular mass, it allows the identification of proteins that might have a role in lung fibrogenesis. Further longitudinal studies on larger cohorts of patients will be necessary to assess their usefulness as predictive markers of disease.

Influence of Aging on Some Specific and Nonspecific Mechanisms of the Host Defense System in 146 Healthy Subjects

The influence of aging on some parameters of systemic host defense mechanisms, i.e. white cell counts, lymphocyte subpopulations, delayed-type hypersensitivity (DTH), polymorphonuclear and mononuclear leukocyte functions, was evaluated. One hundred and forty-six healthy volunteers (60 men and 86 women), aged 25-100 years, were enrolled. None of the subjects had taken any drug in the month before the study. Subjects were divided into three age groups: 25-45, 46-65 and 66-100 years. Groups were comparable in size, and sex distribution was similar throughout all age groups. Elderly people were 51 healthy volunteers between the ages of 66 and 100 years (mean age 79.2). Younger people were 41 subjects between the ages of 46 and 65 years (mean age 54.3) and 53 between the ages of 25 and 45 years (mean age 32.7). As for the comparison between sexes, no significant differences in the values of the studied parameters were found between males and females (p > 0.05). Only quantitative DTH data, i.e. the number of antigens producing positive reactions and the score (sum of positive reaction diameters), were significantly (p < 0.05) reduced in responsive females when compared to males. Aging did not affect white cell counts, lymphocyte subsets and many phagocytic functions, i.e. phagocytosis frequency and index, nitroblue tetrazolium reduction, superoxide production, microbicidal activity against bacteria and yeasts. A significant decrease (p < 0.05) in the chemotactic response to serum-derived chemotactic factors was observed in aged people in comparison to younger subjects. Anergy was more frequent in older (about 29%) than in younger (5-9.4%) healthy volunteers.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Evaluation of Systemic Host Defense Mechanisms in Chronic Bronchitis

Seventy-six chronic bronchitis patients were studied in order to determine the possible presence of disorders in their systemic defense mechanisms. No significant difference in lymphocyte subsets, in serum immunoglobulin and complement component (C3 and C4) levels was found in chronic bronchitis patients compared to normal adult controls. Skin tests for delayed hypersensitivity revealed a high frequency (39%) of hypoergic patients (with 1-2 positive reactions) in comparison to normal subjects. Altered values of many functional properties of both neutrophils and monocytes were demonstrated. The percentage of patients with intermediate (between 1 and 2 SD below the mean of controls) and defective (lower than 1.96 SD) values of chemotaxis, phagocytosis index and Candida killing was about 50%. Phagocytosis frequency and nitroblue tetrazolium reduction frequency were less frequently impaired.

Diazepam-binding inhibitor-derived peptides induce intracellular calcium changes and modulate human neutrophil function.

We studied the effects of two diazepambinding inhibitor (DBI)‐derived peptides, triakonta‐tetraneuropeptide (DBI 17–50, TTN) and eiksoneuropeptide (DBI 51–70, ENP), on cytosolic free Ca2+ concentrations ([Ca2+]i), chemotaxis, superoxide anion (O) generation, and phagocytosis in human neutrophils. Both TTN and ENP induced a rapid and transient rise of [Ca2+]i. The effect of TTN depended on the presence of extracellular Ca2+, whereas the effect of ENP also persisted after extracellular Ca2+ chelation. TTN induced neutrophil chemotaxis, stimulated O generation, and enhanced phagocytosis. ENP did not affect cell migration and oxidative metabolism but enhanced phagocytosis. Both peptides modulated N‐formylmethionyl‐leucyl‐phenylalanine‐ and phorbol myristate acetate‐induced O generation. Because neutrophils express benzodiazepine receptors of the peripheral type (pBRs) and DBI‐derived peptides may interact with such receptors, we investigated the possible role of pBRs in TTN‐ or ENP‐induced effects. The synthetic pBR ligand RO 5‐4864 increased [Ca2+]i through extracellular Ca2+ influx and this effect was prevented by the pBR antagonist PK‐11195. RO 5‐4864, however, was ineffective on neutrophil migration and O generation and only slightly affected phagocytosis. Moreover, PK‐11195 delayed the [Ca2+]i rise induced by TTN but did not significantly affect its extent, and had no effect on the [Ca2+]i rise induced by ENP. We conclude that DBI‐derived peptides induce [Ca2+]i changes and modulate neutrophil function mainly through pBR‐independent pathways. In view of the wide cell and tissue distribution of DBI in the brain and in peripheral organs, modulation of neutrophil function by DBI‐derived peptides may be relevant for both the neuroimmune network and the development and regulation of the inflammatory processes. J. Leukoc. Biol. 67:637–643; 2000.

Diazepam Stimulates Migration and Phagocytosis of Human Neutrophils: Possible Contribution of Peripheral-Type Benzodiazepine Receptors and Intracellular Calcium

In isolated human neutrophils, diazepam (10 nmol/l to 10 µmol/l) concentration-dependently increased migration and phagocytosis. Diazepam-induced migration and phagocytosis were inhibited by the peripheral benzodiazepine receptor (PBR) antagonist PK11195 (10 µmol/l). The PBR agonist Ro5-4864 (10 nmol/l to 10 µmol/l) did not affect migration but slightly enhanced phagocytosis, while clonazepam, which binds to the central-type benzodiazepine receptors but has no affinity for PBRs, was ineffective on both parameters up to 10 µmol/l. Phagocytosis induced by diazepam or Ro5-4864 was inhibited by the Ca2+ channel blocker L-verapamil (10 µmol/l), which however did not affect the action of diazepam on migration. Competition binding experiments performed by fluorescent staining of PBRs showed that diazepam directly interacts with PBRs on human neutrophils. Both diazepam and Ro5-4864 (10 nmol/l to 10 µmol/l) induced a rise of intracellular free Ca2+ concentrations ([Ca2+]i), which was inhibited by PK11195 (10 µmol/l) and L-verapamil (10 µmol/l) and prevented by extracellular Ca2+ chelation with EGTA (5 mmol/l). In conclusion, experimental evidence indicates that in human neutrophils diazepam stimulates both migration and phagocytosis through activation of PBRs. Diazepam-induced [Ca2+]i changes depend on a PBR-operated, L-verapamil-sensitive increase in the plasma membrane permeability and subsequent extracellular Ca2+ entry, and contribute to diazepam-induced phagocytosis. On the contrary, the effect of diazepam on migration seems to occur through Ca2+-independent mechanisms.

Acute Chlamydia pneumoniae and Mycoplasma pneumoniae Infections in Community-Acquired Pneumonia and Exacerbations of COPD or Asthma: Therapeutic Considerations

Abstract Rates of acute Chlamydia pneumoniae and Mycoplasma pneumoniae infections were determined in 115 adults hospitalized for community-acquired pneumonia (CAP), purulent exacerbations of COPD and acute exacerbations of bronchial asthma, by means of serology and molecular methods. Results were compared with those obtained in a matched control group. Common respiratory pathogens were isolated by cultures in 22.5% and 22.2% of CAP and exacerbated COPD patients, respectively. Cultures from exacerbated asthma patients were always negative. Serological and molecular evidence of current C. pneumoniae infection was obtained in 10.0%, 8.9% and 3.3% of CAP, COPD and asthma cases. The corresponding rates of acute M. pneumoniae infection were 17.5%, 6.7% and 3.3%, respectively. Finally, no difference was found between typical and atypical pathogen rates. These findings highlight the importance of taking into account C. pneumoniae and M. pneumoniae infections in guiding the choice of empirical antibacterial treatment for CAP and purulent exacerbations of COPD.

In vitro and ex vivo Enhancement of Nonspecific Phagocytosis by Cefodizime

The in vitro and ex vivo effects of cefodizime on some functional activities of both human neutrophils and monocytes were studied. In vitro experiments were performed with antibiotic concentrations ranging from 1 to 200 micrograms/ml. For the ex vivo study, 7 adult healthy controls were treated intravenously with 4 g/day of cefodizime for 6 days. We found that the drug modulated phagocytosis frequency and index when nonopsonized zymosan and heat-killed Candida albicans were used as phagocytic challenge both in vitro (from 25 micrograms/ml) and ex vivo 12 h after the last administration of cefodizime. No effect on the other phagocyte functional parameters was shown. The in vitro enhancement of nonspecific phagocytosis was demonstrated both in the presence of cefodizime and when phagocytes and particles were separately incubated with the drug.

Effect of beta-lactam antibiotics on migration and bactericidal activity of human phagocytes.

The effect of beta-lactam antibiotics upon some functions of human phagocytes was examined. Penicillins (carbenicillin and piperacillin), thienamycin, and cephalosporins (cefotetan, ceftazidime, and moxalactam) had no effect on either the random or directional migration or on the bactericidal activity of neutrophils and monocytes against Staphylococcus aureus. On the contrary, cefoperazone at therapeutic levels was shown to inhibit neutrophil chemotaxis.