Post-treatment changes of six cytokines in active pulmonary tuberculosis: differences between patients with stable or increased fibrosis.

SETTING: Little information is available regarding the relationship between the fibrotic evolution of pulmonary tuberculosis (PTB) and cytokine levels in human bronchoalveolar lavage fluid (BALF) and serum. OBJECTIVE: To evaluate correlations between profibrotic cytokine levels and post-treatment lung fibrotic evolution. DESIGN: BALF and serum amounts of pro- or anti-inflammatory cytokines were obtained by ELISA before and 6 months after the start of anti-tuberculosis chemotherapy in 13 subjects with PTB. BALF levels were recalculated as ELF (epithelial lining fluid) levels by the urea method. High resolution computed tomography (HRCT) of both lungs was performed at the same time. RESULTS: When comparing pre- and post-treatment radiological data, the scores for 2-10 mm nodules, consolidation and fibrosis presented significant differences (P < 0.05). Concomitantly, pre-treatment vs. 6 month concentrations of ELF IFN-gamma and TNF-alpha were decreased (P < 0.05), while those of IL-4 and IL-10 were increased (P < 0.012). At serum level, IFN-gamma decreased, as did TNF-alpha, TGF-beta1 and PDGF-BB (P < 0.05). When the patients were subdivided into two groups, 1) stable or 2) increasing HRCT fibrosis score, significant increases in the second group were observed for ELF/ serum values of TGF-beta1 and ELF PDGF-BB (P < 0.05) at 6 months post-treatment. Only serum TGF-beta1 values were significantly associated with the same group before treatment.

Expression of CCR5 is increased in human monocyte-derived macrophages and alveolar macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis infection.

Human immunodeficiency virus (HIV) replicates more efficiently in Mycobacterium tuberculosis (MTB)-infected macrophages than in uninfected controls. We investigated whether this may be partly explained by changes in expression of CCR5 in the course of mycobacterial infection, as this molecule has been shown to be a coreceptor for HIV entry. Since the lung is the preferential organ of HIV replication in the course of tuberculosis, we preliminarily analyzed beta-chemokine receptor expression in alveolar macrophages from patients with active tuberculosis, using flow cytometry based on an MIP-1alpha ligand-biotin/avidin-FITC detection system. Increased MIP-1alpha receptor (MIP-1alphaR) expression in alveolar macrophages from infected patients was observed whereas no detectable expression could be revealed in uninfected controls. Since MIP-la can also bind CCR1 and CCR4, the presence of CCR5 mRNA was investigated in bronchoalveolar lavage (BAL) cells and detected in alveolar macrophages from tuberculosis patients only. The study was then extended to in vitro MTB-infected macrophages. Monocyte-derived macrophages (MDMs) were left to differentiate for 7 days before MTB H37Rv infection, and CCR5 expression was monitored, by using a specific monoclonal antibody, on days 1, 6, and 11 after infection. Increased CCR5 expression in MTB-infected macrophages was observed, with a peak on day 6 (64% in MTB-infected versus 33% in control cultures) and a decrease by day 11 (25% in MTB infected versus 13% in control cultures). These results show that CCR5 expression is enhanced in the course of in vitro MTB infection and during active pulmonary tuberculosis.

Aerosolized interferon-alpha treatment in patients with multi-drug-resistant pulmonary tuberculosis.

Multi-drug-resistant tuberculosis (MDR-TB) has emerged as an obstacle to the control of tuberculosis. Recent data however, suggest that interferon-(IFN)-gamma and IFN-alpha may improve disease evolution in subjects affected with pulmonary tuberculosis caused by multi-resistant (IFN-gamma) and sensitive (IFN-alpha) strains. The mechanisms involved are not known, even though it has been reported that IFN-gamma-secreting CD4+ Th cells may possess antitubercular effects. In addition, IFN-alpha can induce IFN-gamma secretion by CD4+ Th cells, and both types of IFN may stimulate macrophage activities. The aim of this study was to explore the possibility that aerosolized IFN-alpha, administered concomitantly with conventional antitubercular chemotherapy, may improve the course of pulmonary tuberculosis. After six months of directly observed therapy (DOT), seven patients who were non-responders to a second line antitubercular therapy were given an IFN-alpha aerosol (3 MU, three times a week) for two months as adjunctive therapy. All strains were resistant to at least two first-line drugs. After IFN-alpha administration, the patients were followed up for a further six months with the same DOT. Sputum samples were collected monthly during the study period, with the exception of the IFN-alpha administration period, when the observations were performed weekly. High resolution computed tomography (HRCT) chest scans were performed before and after IFN-alpha inhalations. The analysis of the results showed that the mean number of Mycobacterium tuberculosis (Mt) had remained statistically unchanged (p = 0.80) during the first 6 months of DOT. During the following 2 months of IFN-alpha administration, 5 patients became negative (p = 0.02). After the end of treatment a progressive increase in Mt number was observed (p = 0. 02). Sputum cultures remained positive for all patients throughout the study period, although a significant decrease (p = 0.02) in the colony number per culture was observed after adjunctive treatment with IFN-alpha. After stopping administration of IFN-alpha, a significant increase (p = 0.03) in the colony number per culture was noted as well as in Mt numbers. HRCT scans were slightly improved in all patients. These preliminary data suggest that aerosolized IFN-alpha may be a promising adjunctive therapy for patients with MDR-TB. Optimal doses and schedules however, require further studies.

Different cytokine production and effector/memory dynamics of alpha beta+ or gamma delta+ T-cell subsets in the peripheral blood of patients with active pulmonary tuberculosis.

Immunity to M.tuberculosis (MTB) infection consists of interactions between various T-cell subsets that control the infection and prevent further reactivation. We analysed the effector/memory T-cell dynamics and cytokines production in the peripheral blood of patients with pulmonary tuberculosis (TB). We observed that the frequency of CD4+ T-cell effectors was significantly increased during active TB, confirming a major role of this T-cell subset in TB immunity. Pre-terminally differentiated CD8+ T-lymphocytes were increased in the peripheral blood as well. In contrast, we observed a reduced number of effector mycobacteria-reactive gammadelta+ T-lymphocytes with a specific defects in reacting to mycobacterial nonpeptidic antigens, suggesting that this innate response is rapidly lost during TB infection. Nevertheless, the frequency of gammadelta+ T-cells effectors in TB patients was higher than the alphabeta+ T-cell response to peptide from MTB-ESAT-6 protein and quantitatively similar to PPD reactivity. Thus, alphabeta+ and gammadelta+ T-cell differentiation and function are differently triggered by active TB infection.

Detection of allergens in plantain (Plantago lanceolata) pollen.

BACKGROUND: Allergens in Plantago lanceolata have not been characterized yet. The objective was to characterize some plantain-pollen allergens and to investigate the cross-reactivity between plantain and grass pollens. METHODS: Sera from four patients monosensitive to plantain pollen and from eight grass-pollen-allergic patients showing strong skin reactivity to plantain pollen in the skin prick test (SPT) underwent immunoblot analysis with both Plantago and grass mix extract. Moreover, immunoblot inhibition experiments were done with grass mix extract as inhibitor. RESULTS: All four sera from plantain-allergic patients reacted to two distinct bands at 17 and 19 kDa, and 2/4 sera showed further reactivity to a 40-kDa protein, which in one case represented the most prominent IgE-binding allergen. Plantain-monosensitive subjects did not show any reactivity to grass-pollen extract, and preabsorption of their sera with grass-pollen extract did not cause any loss of reactivity to plantain pollen. Sera from all eight grass-pollen-allergic controls reacted to a 30-kDa protein in plantain pollen, and some sera showed cross-reactivity to higher and lower molecular-weight structures as well. In all cases, plantain reactivity was totally abolished by preabsorption of sera with grass-pollen extract. A preliminary investigation by immunoblot showed that polyclonal IgG anti-Phl p 5 (but not polyclonal Phl p 1) from rabbit reacted to a 30-kDa protein in plantain pollen. CONCLUSIONS: Three specific allergens (of 17, 19, and 40 kDa, respectively) have been detected in plantain pollen. Further studies on a larger number of patients will determine whether these proteins may be considered major allergens. Cross-reactivity between grass and plantain pollen is mainly caused by a 30-kDa protein in plantain pollen. Group 5 grass-pollen allergen is probably responsible for most grass/plantain cross-reactivity.

Allergy to nonspecific lipid transfer proteins in Rosaceae: a comparative study of different in vivo diagnostic methods.

BACKGROUND: Lipid transfer proteins (LTPs) are the major allergens in patients sensitive to Rosaceae (apple, peach, apricot, cherry, plum, and pear) who are not allergic to birch pollen. OBJECTIVE: The purpose of this study was to find a sensitive, specific, and relatively easy method for detection of LTP-sensitive patients. METHODS: We studied 36 persons who experienced oral allergy syndrome after the ingestion of fruits in the family Rosaceae. This study cohort was divided into two groups: 18 without allergy to birch pollen (patients) and 18 with birch pollen allergy (control subjects). All were tested by skin prick tests (SPTs) with fresh Golden Delicious apple, fresh peach, and extracts of peel and pulp from both fruits. Their specific IgE reactivities against peach peel extract were further investigated by immunoblot analysis. RESULTS: All 18 subjects in the control group showed strongly positive skin reactions with both fresh apple and fresh peach, whereas no skin reactivity was found with extracts from peach peel, peach pulp, or apple pulp. Extract of apple peel produced positive skin reactions in 17 of 18 control subjects; however, the wheals were generally smaller than those induced by fresh fruits. Immunoblot analysis showed no reactivity for peach peel extract. In contrast, the SPTs with fresh fruits showed that some of the 18 patients had strongly positive reactions, but others had weak reactions or negative responses. Further, in a high proportion of the patients, consecutive SPT with fresh apple yielded inconsistent results. In all patients, SPTs with extracts from apple pulp and peach pulp were negative, whereas SPTs with peel extracts were strongly positive in all patients. In most patients, the wheal area induced by SPT with peel extracts was larger than that induced by SPTs with fresh fruits. Immunoblot analysis showed that serum specimens from all 18 patients reacted with a 10-kD protein in peach peel. This is the molecular mass of LTPs. CONCLUSIONS: In birch pollen-allergic patients, the SPTs with fresh foods still remains the most reliable method of diagnosing vegetable food hypersensitivity. In contrast, in patients not allergic to birch pollen, the most reliable strategy for detection of patients sensitive to LTPs is skin prick testing with properly prepared fruit peel extracts. The loss of Bet v 1- and Bet v 2-like structures, which probably occurs during extraction, may facilitate immediate identification of the relevant allergen.

Minimal dose of aerosolized interferon-alpha in human subjects: biological consequences and side-effects.

Interferon-alpha (IFN-alpha) is a leucocyte-derived cytokine with pleiotropic effects on the cells of the immune system, including the ability to promote viral and microbial killing. This study was designed to evaluate the biologically active dosage of aerosolized lymphoblastoid IFN-alpha, in normal subjects and patients with chronic bronchitis, using serum 2'-5' oligoadenylate synthetase (OAS) as a marker of IFN-alpha activity. Three groups of subjects were included: two healthy groups and one of patients with chronic bronchitis. Group A (controls, n = 5) was studied in order to determine the minimal IFN-alpha dose able to induce biological effects without side-effects. IFN-alpha was given in a dose escalation trial including 0, 0.3 x 10(6), 1.0 x 10(6) and 3.0 x 10(6) IU.day-1 (5 day administration). Only the administration of 3.0 x 10(6) IU.day-1 of IFN-alpha induced a significant biological activity, increasing serum levels of OAS. Group B (controls, n = 5) and C (chronic bronchitis, n = 5) were given 3.0 x 10(6) IU.day-1 (10 day administration) in order to study serum, bronchoalveolar lavage fluid (BALF) and BALF cell modifications, after treatment. OAS serum levels and nitroblue tetrazolium (NBT) reduction tests, the latter used as a measure of phagocyte cell activity, increased both in normal subjects and in patients with chronic bronchitis. No significant change of serum IFN-alpha levels was found. It is concluded that aerosolized IFN-alpha administration to the lung is well-tolerated at biologically active doses. The activity can be monitored by quantifying OAS serum levels through a simple blood test.

Percutaneous transhepatic sphincterotomy of the major papilla and stone extraction without endoscopic control.

Obstructive jaundice due to an impacted stone in the common bile duct (CBD) was seen in a patient who had previously undergone Billroth II gastric resection and cholecystectomy. Surgical and endoscopic approaches to the common bile duct failed owing to pericholedochal adhesions and the excessive length of the duodenal loop. The patient was therefore treated percutaneously (sphincterotomy and stone extraction) without endoscopic control.

Effects of aerosolized interferon-alpha in patients with pulmonary tuberculosis.

Interferon-alpha (IFN-alpha) is a cytokine exerting pleiotropic activities, including antimicrobial effects, especially directed against intracellular infectious bacteria. It may be administered by aerosol to reach the lower respiratory tract without systemic side effects. The aim of the study reported here was the evaluation of aerosolized IFN-alpha treatment (3 MU/dose, given three times a week; total study dose: 72 MU/2 mo) in combination with conventional antimycobacterial therapy in patients with pulmonary tuberculosis. Two groups of 10 patients each were compared before and after 2 mo of conventional antituberculous chemotherapy with or without inhaled IFN-alpha. Several biologic (bronchoalveolar lavage fluid [BALF] cellularity, Mycobacterium tuberculosis [MT] number in sputum), biochemical (BALF concentrations of 10 cytokines, BALF IFN-alpha receptor levels), and clinical (fever, vital signs, high-resolution computed tomography [HRCT] images) measures were made in these patients at the time of their enrollment and at the end of the observation period of the study. Fever, MT number in sputum, and abnormalities in HRCT images showed significantly earlier resolution in the IFN-alpha-treated group, together with a more significant decrease in BALF interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) concentrations and significantly greater pre- versus posttreatment variations in IL-2 and IFN-gamma. These data, taken together, suggest that IFN-alpha administration may favorably affect the evolution of pulmonary tuberculosis when combined with antimycobacterial therapy.

Cytokine levels correlate with a radiologic score in active pulmonary tuberculosis.

Pulmonary tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. This microorganism is capable of inducing a delayed hypersensitivity reaction in the lung, with subsequent expression of the disease. This reaction depends on the presence of different cytokines that exert specific functions. The aim of this study was to evaluate the presence and the concentrations of nine different modulators in bronchoalveolar lavage fluid (BALF). For this purpose, 15 patients with active pulmonary tuberculosis were enrolled at the time of diagnosis, prior to institution of antituberculous therapy. All the patients demonstrated M. tuberculosis in the sputum, and their disease extention was defined by high-resolution computed tomography (HRCT) using a score which included the presence of six findings: miliary nodules, nodules < 10 mm, consolidation, ground glass, cavity and bronchial wall thickening. This score was more sensitive than an equivalent score calculated on the basis of chest radiology. HRCT score was calculated for each area of the two lungs in order to define the more and the less affected lung for each patient. The bronchoalveolar lavage (BAL) was performed in the more affected area for each lung. The HRCT total score for each washed area ranged between 1 and 15, and showed more significant differences between the more and less affected lungs (p = 0.0004) than those obtained with the individual radiologic findings (p ranged between 0.60 and 0. 004). The BAL concentrations of the nine cytokines evaluated for the more and less affected lungs were compared: interleukin-6 (IL-6), IL-8, IL-12, tumor necrosis factor-alpha (TNF-alpha), and interferon gamma (IFN-gamma) showed significant differences (p ranged between 0. 016 and 0.0007). In addition, each cytokine concentration was correlated with the HRCT score. Significant correlations were found with IL-12, IL-6, IL-8, IL-2, and TNF-alpha. The correlations between cytokines and HRCT total score were better than those observed with the individual radiologic findings. A correlation matrix for the different cytokines evaluated one against each other, has also been added to show common behavior of these modulators. A similar analysis was also performed for the radiologic abnormalities.

Presenilin-1 regulates the neuronal threshold to excitotoxicity both physiologically and pathologically.

A direct pathophysiological role of Familial Alzheimer's Disease (FAD)-associated Presenilin 1 (PS1) mutations in neuronal vulnerability remains a controversial matter. We evaluated the relationship between PS1 and excitotoxicity in four different experimental models of neurotoxicity by using primary neurons from (i) transgenic (tg) mice overexpressing a human FAD-linked PS1 variant (L286V mutation), (ii) tg mice overexpressing human wild-type (wt) PS1, (iii) PS1 knockout mice, and (iv) wt mice in which PS1 gene expression was knocked down by antisense treatment. We found that primary neurons overexpressing mutated PS1 showed an increased vulnerability to both excitotoxic and hypoxic-hypoglycemic damage when compared with neurons obtained from either mice overexpressing human wt PS1 or in wt mice. In addition, reduced excitotoxic damage was obtained in neurons in which PS1 expression was absent or diminished. Data obtained in in vivo experimental models of excitotoxicity partially supported the in vitro observations. Accelerated neuronal death was demonstrated in the hippocampus of mice overexpressing mutated PS1 after peripheral administration of kainic acid in comparison with wt animals. However, measurement of the infarct volume after middle cerebral artery occlusion did not show significant difference between the two animal groups. The results altogether suggest that expression of FAD-linked PS1 variants increases the vulnerability of neurons to a specific type of damage in which excitotoxicity plays a relevant role. In addition, they support the view that reduction of endogenous PS1 expression results in neuroprotection.

Effect of octreotide on sphincter of Oddi motility in patients with acute recurrent pancreatitis: a manometric study.

Sphincter of Oddi dysfunction has been reported as a cause of acute idiopathic recurrent pancreatitis (IRP). Octreotide, a long-acting somatostatin analogue, is an antisecretory drug used in the treatment and prevention of acute pancreatitis. Its action on sphincter of Oddi motility is controversial and no data are available for IRP patients. The aim of this study was to assess sphincter of Oddi motor response to acute administration of octreotide in patients with past attacks of acute pancreatitis without identification of any evident aetiological factor. Six patients (four male, two female; mean age +/-SD, 38.8+/-9 years) suffering from acute pancreatitis for at least 3 months before the examination were submitted to sphincter of Oddi manometry. After a basal recording lasting at least 2 min, octreotide, 0.05 mg i.v., was administered and the recording repeated. Intraduodenal pressure was taken as the zero reference and the basal sphincter of Oddi pressure and amplitude and frequency of phasic contractions were calculated before and after octreotide administration. No significant pre- vs post-octreotide differences were observed in basal pressure (41.9+/-24 vs 47.5+/-33 mm Hg, respectively) or in amplitude of phasic contractions (164.6+/-33 vs 170.8+/-18 mm Hg). With a latency of about 1 min, octreotide administration caused a high-frequency phasic activity in all cases (mean frequency, 5.5+/-2.2 contractions/min before and 9.8+/-2 after octreotide; P < 0.04). After the procedure acute pancreatitis (prolonged abdominal pain and serum amylase levels more than three-fold the normal values) developed in five patients. In conclusion, our data suggest that acute administration of octreotide may induce tachyoddia and thus a rise in sphincter of Oddi pressure, with possible impairment of biliary-pancreatic outflow.

Mycobacterium tuberculosis-induced apoptosis in monocytes/macrophages: early membrane modifications and intracellular mycobacterial viability.

Apoptosis has been observed in monocytes/macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis (MTB) infection. In order to define the early events of MTB-induced apoptosis, membrane CD14 expression and the exposure of Annexin V-binding sites in MTB-infected monocytes/macrophages have been monitored. Moreover, the role of MTB-induced apoptosis was further analyzed in vitro in terms of mycobacterial viability. Results show that monocyte/macrophage apoptosis is a very early event that is strictly dependent on the MTB amount, and this apoptosis is associated with a selective down-regulation of surface CD14 expression. Furthermore, no statistically significant decrease in mycobacterial viability was observed, which indicates that the apoptotic pathway triggered by high doses of MTB is associated with parasite survival rather than with killing of the parasite.