COPD treatment: real life and experimental effects on peripheral NK cells, their receptors expression and their IFN-γ secretion.

A role in pulmonary immunity has been ascribed to Natural Killer (NK) cells and several in vitro studies have shown a corticosteroid-induced inhibition of NK cells mediated cytotoxicity. Several clinical trials on chronic obstructive pulmonary disease (COPD) have suggested a relationship between COPD treatment and occurrence of respiratory infections. Aims of our study were to investigate if real life COPD treatment affects peripheral blood NK cells total count and their receptors expression and to assess if different doses of formoterol and budesonide, administered alone or in combination, are able to modulate the surface expression of activating (NKp30, NKp44, NKp46 and NKG2D) and inhibitory (KIR2DL2/L3, KIR3DL1 and NKG2A) receptors on peripheral blood NK cells of COPD patients. Moreover, we evaluated the potential effect of treatment with budesonide and/or formoterol on IFN-γ secretion in vitro. NK cells were isolated from peripheral blood of 7 healthy volunteers, 9 chronic bronchitis (CB) and 11 COPD patients. Total NK cells count and activating and inhibitory receptors expression were evaluated. NK cells were cultured for 20h in 96-well plates with IL-2 (100IU/ml)+IL-12 (2.5ng/ml), with or without budesonide (Bud; 1 and 0.01µM) and formoterol (For; 30 and 0.3nM) alone or in combination. Cells were analyzed by flow cytometry and IFN-γ was measured in cell supernatants by ELISA test. No difference between real life treated COPD, CB and healthy subjects was found concerning NK total count and NK cell receptors expression. When cells were stimulated over night with cytokines and treated with drugs, only NKG2D receptor was modulated. Its expression was significantly downregulated by budesonide alone and in combination with formoterol in COPD patients. IFN-γ production induced by stimulation with IL-2+IL-12 was decreased in a highly significant way (p<0.01) by all treatments in all groups. Even if in vitro experiments with budesonide, alone or in combination with formoterol, showed a modulation of NKG2D receptor expression and IFN-γ production, our ex vivo results show that real life LABA and ICS treatment does not influence peripheral NK cells count and their receptors phenotype.

Omalizumab modulates bronchial reticular basement membrane thickness and eosinophil infiltration in severe persistent allergic asthma patients.

Severe persistent asthma causes a substantial morbidity and mortality burden and is frequently not well controlled, despite intensive guideline-based therapy. The unique monoclonal antibody approved for patients with severe allergic asthma is omalizumab: a recombinant humanised murine against IgE antibodies. The aim of the present study is to investigate the effect of long-term anti-IgE on the thickening of the reticular basement membrane (RBM) and eosinophil infiltration in bronchial biopsies from patients with severe persistent allergic asthma. Biopsies were obtained from 11 patients with severe persistent allergic asthma before and after (12 months) treatment with omalizumab. RBM thickness and eosinophils were measured by using light microscope image analysis. A significant mean reduction in RBM thickness and eosinophil infiltration were measured after one-year omalizumab treatment. No correlation between eosinophil reduction and RBM thickness reduction was found. No correlation between each of the previous two parameters and clinical parameters was detected. In conclusion, our study showed that a substantial proportion of severe asthmatics reduced the original bronchial RBM thickness and eosinophil infiltration after one-year treatment with anti-IgE, thus emphasizing the possible role of omalizumab in affecting airway remodeling in severe persistent allergic asthma.

Salbutamol: how does it enter smooth muscle cells?

Polyspecific organic cation transporters (OCTs) in human cell membranes are involved in the uptake, distribution and excretion of cationic compounds. Although their relevance to drug disposition in the liver, small intestine and kidney has been investigated previously, less is known about the influence of these transporters on the pharmacokinetics and pharmacodynamics of inhaled drugs. Drugs that are commonly administered by inhalation for the treatment of respiratory diseases, such as glucocorticoids and cationic ß(2)-agonists, might interact with several of these transporters, which are strongly expressed on the surfaces of airway epithelial cells. We evaluated the expression of OCT3 and measured the in vitro uptake of the short-acting ß(2)-agonist salbutamol (SALB), alone or in combination with corticosterone (CS) and beclomethasone dipropionate (BDP), by bronchial smooth muscle cells. Our results showed that these cells express the OCT3 transporter and that SALB enters the cell in a transporter-independent fashion. Moreover, CS and BDP have different activities on SALB transport inside the cell. CS increases SALB transport and BDP decreases SALB transport, although neither of these effects are statistically significant. A better understanding of these mechanisms might lead to the improved treatment of airway diseases.

Anti-proliferative and anti-remodelling effect of beclomethasone dipropionate, formoterol and salbutamol alone or in combination in primary human bronchial fibroblasts.

BACKGROUND: Bronchial asthma is characterized by lower airway inflammation and remodelling. Anti-inflammatory treatment with inhaled corticosteroids (ICS) provides the mainstay of asthma therapy together with bronchodilation induced by short- and long-acting inhaled beta(2)-agonists. Lower airway fibroblasts may play a critical role in airway inflammation and remodelling, suggesting that they might represent an important target for the major anti-asthmatic drugs. The aim of our study was to investigate the effects of beclomethasone dipropionate (BDP), salbutamol and formoterol either alone or in combination on in vitro cultures of human bronchial fibroblasts. METHODS: Fibroblasts were cultured in the presence of pro-inflammatory and proliferative stimuli, BDP, salbutamol and formoterol. The effects of drugs on cell proliferation were ascertained by (3)H-thymidine incorporation. CD90 and CD44 expression were detected by flow cytometry and fibronectin secretion using the enzyme-linked immunosorbent assay technique. RESULTS: This study showed that BDP alone has significant anti-proliferative effects on lung fibroblasts treated with basic fibroblast growth factor and the combination of BDP with formoterol or salbutamol strengthen these effects. Short-acting beta(2)-agonist (SABA) or long-acting beta(2)-agonist (LABA) by themselves did not show any significant effect on the different cultures. CD44 and CD90 expression and fibronectin production were modulated by pro-inflammatory and proliferative stimuli; the addition of the drugs brought them back near to the basal level. CONCLUSIONS: From this in vitro study, we can conclude that BDP, when combined with salbutamol or formoterol, exhibits enhanced anti-remodelling activity in bronchial fibroblasts, providing new insights on the additive effects of ICS and SABAs and LABAs for asthma therapy.

Effect of statins on fibroblasts from human nasal polyps and turbinates.

BACKGROUND: Statins are serum cholesterol-lowering agents used for the prevention and treatment of atherosclerotic vascular disease. There is, however, growing evidence that statins have immunomodulatory and anti-inflammatory activities and may prove invaluable in the treatment of immunological and inflammatory disorders. OBJECTIVE: On these basis we evaluated the effect of statins on the proliferation of fibroblasts derived from human nasal polyps and turbinates and determined their ability to modulate airway remodelling. METHODS: Fluvastatin (0.01-0.1-1 microM), Atorvastatin (0.1-1-10 microM) and Simvastatin (0.1-1-10 microM) were tested on cultured fibroblasts derived from human nasal polyps and turbinates stimulated or not with Fibroblast Growth Factor beta (10 ng/ml). All cultures were treated with 3H-Thymidine (1 microCi/ml) to test cell proliferation. RESULTS: Our results show that proliferation of turbinate-derived fibroblasts is significantly inhibited by the three statins. Fluvastatin is already effective at the lowest dose (0.01 microM), whereas Atorvastatin and Simvastatin act at the plasmatic peak concentration (1 microM). No significant effect was found on fibroblasts derived from nasal polyps, except for Simvastatin which was effective after 144 hours of stimulation. CONCLUSIONS: These drugs show a remarkable antiprolhferative effect and their different outcome depending on the different kind of fibroblasts in vitro is prompting news in the studies about statin use for the treatment of chronic inflammatory diseases.

Levocetirizine in persistent allergic rhinitis and asthma: effects on symptoms, quality of life and inflammatory parameters.

Background Levocetirizine (LCZ) has been shown to be effective in allergic rhinitis. We evaluated its clinical efficacy, antinflammatory actions and its effects on quality of life (QoL) with a specific instrument in the asthma-rhinitis comorbidity. Methods Fifty adult patients with persistent rhinitis with/without asthma were enrolled. After a 1-week run-in for baseline evaluation, they were randomized to LCZ or placebo for 8 weeks. Cromolyn and salbutamol were permitted on demand. Rhinoconjunctivitis and asthma symptoms were evaluated by diary cards. QoL was assessed by the specific Rhinasthma questionnaire and the generic SF-36 at different time-points. Nasal scrapings and lavages were also performed for inflammatory cell count and mediator assessment. Results Ten patients dropped out for unrelated reasons and the remaining completed the study with no side-effect. Symptoms began to decrease in the active group at the second week of treatment when the difference with the placebo group became significant (0.05) and so remained until the end of the trial. Starting from 2 weeks of therapy, there was a significant decrease vs. baseline in all the four components of the Rhinasthma questionnaire only in the active group. The intergroup comparison became significant (P<0.05) at 4 weeks. The SF-36 detected only sporadic differences between groups. Eosinophils and neutrophils in nasal scraping were significantly decreased in the LCZ group vs. baseline at all times. Nasal mediators were under the detection limits and no analysis could be performed. In the active group, only two patients used rescue medications compared with 13 patients in the placebo group. Conclusions LCZ is clinically effective and capable of improving the rhinitis-asthma-related QoL.

[Reduction of the incidence of nosocomial infections after assessing and revising the quality of assistance in a thoracic surgery unit]. / Riduzione del tasso d'incidenza delle infezioni ospedaliere in una U.O. di Chirurgia Toracica mediante verifica e revisione della qualità assistenziale.

An active surveillance for nosocomial infections has been lead in a Thoracic Surgery with the intention, first to point out their frequency and characteristics, and then to outline all the measures to remove the main risk factors checking the results obtained. A prospective incidence study has been promoted in a Thoracic Surgery in the years 2000, 2001, 2002. The analysis has been lead weekly gathering all necessary data from the health records and making laboratory tests to look for microbes growth in the air of Thoracic Surgery Operating Rooms. A nosocomial infections incidence of 13.3% among surgically treated patients has been registered in 2000. Deep surgical site infections were the most frequent localizations, and microbes isolated were Staphylococcus aureus and coagulase negative Staphylococcus with an high oxacillin resistance (70.6%-76.5%). From the observation of the risk factors the sterilization system has been modified and the assistance and environmental protocols have been improved. In the further evaluation period, a global reduction of nosocomial infections incidence (7.1%), of surgical site infections (from 10.1% to 4.5%) (p = 0.007), of Staphylococcus aureus and coagulase negative Staphylococcus isolations have been obtained even if short results in antibiotic resistances have been registered. Thoracic Surgery has to be considered an area at medium-high risk of nosocomial infections. The quite high incidence of nosocomial infections recorded at the beginning of the study in presence of prevalent deep surgical site infections from staphylococci with an high oxacillin resistance compelled to promote corrections. These lead to a remarkable decrease in incidence of nosocomial infections even if the same results can not be reached in antibiotic resistances.

Correlation between meteorological conditions and Parietaria pollen concentration in Alassio, north-west Italy.

The pollen grains in the atmosphere in different geographical areas differ according to the species present, the pollination seasons and pollen grain concentrations, but possibly the greatest contributors to this variability are the meteorological conditions. The aim of our research is to establish a possible correlation between Parietaria pollen concentration and meteorological conditions during the period from 1991 to 1995 in the town of Alassio (north-west Italy). As far as vegetation is concerned, the Mediterranean climatic conditions support the blooming of extensive grasslands in the environment surrounding the town; these grasslands mainly comprise Urticaceae and shrubs. The study demonstrates that the most influential parameters affecting the Urticaceae grain concentration upsurge are the absence of rainfall, a maximum daily temperature of about 21 degrees C, and a diurnal temperature range of about 5 degrees C. Moreover, our aeropalinological study indicates that this last parameter has the greatest influence on Urticaceae pollination. In fact, an increase in diurnal temperature range could be responsible for a dehydration of pollens resulting in a loss in mass. This grain lightness and volatility would ultimately permit atmospheric dispersion if there is a significant wind speed. On the other hand, days with rain or high relative humidity make pollens heavier, preventing them from flying long distances and therefore partially explaining the decline in airbone pollen concentration.

High rate of positive anti-tissue transglutaminase antibodies in chronic liver disease. Role of liver decompensation and of the antigen source.

BACKGROUND: Since the recognition of tissue transglutaminase (tTG) as the target antigen of anti-endomysium antibodies, several ELISA assays using either guinea pig or human recombinant tTG have been developed. The aim of the study was to compare the behaviour of anti-tTG and anti-endomysium antibodies assays in coeliacs and in patients with chronic liver disease. METHODS: 34 patients (24 women, 34.9 +/- 12.5 years) with coeliac disease and 41 with chronic liver disease (14 women, 57 +/- 11.2 years), including 19 cirrhotics, were evaluated for anti-endomysium antibodies by indirect immunofluorescence and for anti-tTG IgA antibodies by ELISA, using guinea pig liver or human recombinant transglutaminase. RESULTS: The prevalences of anti-tTG and anti-endomysium antibodies were 100% in patients with coeliac disease at diagnosis, 75% and 64.3% in patients on a gluten-free diet. All liver disease patients were negative for anti-endomysium antibodies, while 11 (26.8%) were positive for anti-tTG. All these patients had liver cirrhosis and represented 57.9% of all cirrhotics. The presence of anti-tTG was associated with higher Child-Pugh scores. The use of human transglutaminase determined a reduction in the rate of positive results; however, the rate of positive anti-tTG was still 17.1% in all liver disease patients and 31.6% in cirrhotics. CONCLUSIONS: Our data confirm that anti-tTG have a similar sensitivity compared with anti-endomysium antibodies assay in coeliacs. However, a high prevalence of positive anti-tTG results is observed in cirrhotic patients, even when human recombinant tTG is used. The high prevalence of positive results among cirrhotic patients is associated with more advanced liver disease.

Diagnostic role and clinical correlates of anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (p-ANCA) in Italian patients with inflammatory bowel diseases.

BACKGROUND: Anti-Saccharomyces cerevisiae antibodies (ASCA) and perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA) are serological markers associated, respectively, with Crohn's disease and ulcerative colitis, whose clinical significance and possible diagnostic role are still poorly defined. AIMS: (a) To evaluate the sensitivity, specificity and predictive values of isolated and combined ASCA and p-ANCA assays in a large cohort of Italian patients with inflammatory bowel disease (IBD) and (b) to assess whether their presence is associated with particular clinical features of the disease. PATIENTS AND METHODS: Hundred and forty-six IBD patients (93 with Crohn's disease and 53 with ulcerative colitis) and 54 control patients were enrolled in the study. ASCA (IgA and IgG) and p-ANCA were determined by means of enzyme-linked immunosorbent assay and indirect immunofluorescence, respectively. RESULTS: The specificities were excellent for both tests (ASCA in Crohn's disease, 98.1% both for IgA and IgG, and p-ANCA in ulcerative colitis, 92.5%); however, the sensitivities of both tests were low (59.1% for ASCA IgA, 44.1% for ASCA IgG, 39.6% for p-ANCA). ASCA specificity and positive predictive value reached 100% when positivity for both IgA and IgG was present. No significant association was found between the presence of a specific serological marker and patients' clinical features. CONCLUSIONS: This study confirms the low prevalence of p-ANCA observed in ulcerative colitis patients from the Mediterranean area. The low sensitivity of ASCA and p-ANCA, despite their rather high specificity, renders them of little value in the screening of the general population, where the prevalence of IBD is low. However, in our series, a double positivity for ASCA IgA and IgG identifies with certainty the presence of Crohn's disease.

High Rate of Positive Anti-Tissue Transglutaminase Antibodies in Chronic Liver Disease.

BACKGROUND: Since the recognition of tissue transglutaminase (tTG) as the target antigen of anti-endomysium antibodies, several ELISA assays using either guinea pig or human recombinant tTG have been developed. The aim of the study was to compare the behaviour of anti-tTG and anti-endomysium antibodies assays in coeliacs and in patients with chronic liver disease. METHODS: 34 patients (24 women, 34.9 ± 12.5 years) with coeliac disease and 41 with chronic liver disease (14 women, 57 ± 11.2 years), including 19 cirrhotics, were evaluated for anti-endomysium antibodies by indirect immunofluorescence and for anti-tTG IgA antibodies by ELISA, using guinea pig liver or human recombinant transglutaminase. RESULTS: The prevalences of anti-tTG and anti-endomysium antibodies were 100% in patients with coeliac disease at diagnosis, 75% and 64.3% in patients on a gluten-free diet. All liver disease patients were negative for anti-endomysium antibodies, while 11 (26.8%) were positive for anti-tTG. All these patients had liver cirrhosis and represented 57.9% of all cirrhotics. The presence of anti-tTG was associated with higher Child-Pugh scores. The use of human transglutaminase determined a reduction in the rate of positive results; however, the rate of positive anti-tTG was still 17.1% in all liver disease patients and 31.6% in cirrhotics. CONCLUSIONS: Our data confirm that anti-tTG have a similar sensitivity compared with anti-endomysium antibodies assay in coeliacs. However, a high prevalence of positive anti-tTG results is observed in cirrhotic patients, even when human recombinant tTG is used. The high prevalence of positive results among cirrhotic patients is associated with more advanced liver disease.

Structure of chicken plasma retinol-binding protein.

The crystal structure of the specific carrier of retinol (retinol-binding protein, RBP) purified from chicken plasma has been determined (space group P2(1)2(1)2(1), with a=46.06(5) A, b=53.56(6) A, c=73.41(8) A, and one protein molecule in the asymmetric unit). Despite being obtained from a species phylogenetically distant from mammals, chicken holoRBP has an overall structure that closely resembles the previously determined structures of mammalian holoRBPs. The lack in chicken RBP of eight carboxy-terminal amino acid residues characteristic of mammalian RBPs does not significantly affect the protein structure. A distinctive feature of the avian protein is a better definition of the loop 63-67, close to the opening of the beta-barrel cavity accommodating the retinol molecule, which is rather disordered in the structures of mammalian RBPs.

Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein.

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified iota-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282-3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans-retinol form a complex (K(d) approximately 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-A x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.

Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells.

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3, 7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl(-) current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

Modification of transepithelial ion transport in human cultured bronchial epithelial cells by interferon-gamma.

Human bronchial epithelial cells were treated in vitro with interferon-gamma or tumor necrosis factor-alpha to assess their effect on transepithelial ion transport. Short-circuit current measurements revealed that Na(+) absorption was markedly inhibited by interferon-gamma (10-1,000 U/ml). The cystic fibrosis transmembrane conductance regulator was also downregulated by interferon-gamma as evident at the protein level and by the decrease in the cAMP-dependent current. On the other hand, interferon-gamma caused an increase of the current elicited by apical UTP application, which is due to the activity of Ca(2+)-dependent Cl(-) channels. Tumor necrosis factor-alpha caused few changes in ion transport. Transepithelial fluid transport was measured in normal and cystic fibrosis cells. At rest, both types of cells showed an amiloride-sensitive fluid absorption that was inhibited by interferon-gamma but not by tumor necrosis factor-alpha. Our results show that interferon-gamma alters the transepithelial ion transport of cultured bronchial cells. This effect may change the ion composition and/or volume of periciliary fluid.

Structure at 1.44 A resolution of an N-terminally truncated form of the rat serum complement C3d fragment.

Complement component C3 plays a key role in the complement-mediated immune defence, and occupies a central position within the complement cascade system. One of its degradation products, C3dg, was purified from rat serum and crystallised in two different crystal forms as N-terminally truncated fragment. Despite the truncation and the lack of a significant portion of the N-terminus as compared to C3d, the structure of the fragment is highly similar to that of recombinant human C3d (Nagar et al., Science 280 (1998) 1277-1281). Structural details of the reactive site have been obtained, suggesting a possible mode of thioester bond formation between Cys-1010 and Gln-1013 and thioester bond cleavage in the transacylation reaction involving His-1126. The truncation at the N-terminus of C3d leads to the exposure of a surface of the molecule that favours dimerisation, so that in both crystal forms, the fragment is present as a dimer, with monomers related by a two-fold axis.

Crystallization and preliminary X-ray data for the human transthyretin-retinol-binding protein (RBP) complex bound to an anti-RBP Fab.

A macromolecular complex of human transthyretin, human retinol-binding protein and an anti-retinol-binding-protein Fab was crystallized by vapour diffusion in sitting drops. Diffraction from these crystals at cryogenic temperatures was consistent with the space group C222, with cell parameters a = 159.34, b = 222.40 and c = 121.27 A. Crystals diffracted to a resolution limit of 3.36 A using synchrotron radiation. Based on a 2:2:1 stoichiometry for the Fab-retinol-binding-protein-transthyretin complex and the presence of one such complex per asymmetric unit, a reasonable Vm coefficient of 2.74 A3 Da-1 could be estimated.

Structure of pig plasma retinol-binding protein at 1.65 A resolution.

The crystal structure of pig plasma retinol-binding protein (RBP) has been determined at 1.65 A resolution. The space group is P212121, with a = 45.81 (4), b = 53.14 (5), c = 72.97 (8) A and one protein molecule in the asymmetric unit. The structure has been solved using the molecular replacement method and refined with restrained least squares to an R factor of 0.1844 and an Rfree of 0.237 for 18 874 and 1001 independent reflections, respectively. The relatively high resolution structure of pig holoRBP has revealed some new structural details. Moreover, it has provided a description of the binding site for Cd2+, a metal ion which is required for protein crystallization. The hepta-coordination of the RBP-bound cadmium ion involves different residues of two symmetry-related RBP molecules, consistent with the participation of the cation in intermolecular interactions that in turn promote protein crystallization.

Structure of the trigonal crystal form of bovine annexin IV.

The structure of a trigonal crystal form of N-terminally truncated [des-(1-9)] bovine annexin IV, an annexin variant that exhibits the distinctive property of binding both phospholipids and carbohydrates in a Ca2+-dependent manner, has been determined at 3 A (0.3 nm) resolution -space group: R3; cell parameters: a=b=118.560 (8) A and c=82.233 (6) A-. The overall structure of annexin IV, crystallized in the absence of Ca2+ ions, is highly homologous to that of the other known members of the annexin family. The trimeric assembly in the trigonal crystals of annexin IV is quite similar to that found previously in non-isomorphous crystals of human, chicken and rat annexin V and to the subunit arrangement in half of the hexamer of hydra annexin XII. Moreover, it resembles that found in two-dimensional crystals of human annexin V bound to phospholipid monolayers. The propensity of several annexins to generate similar trimeric arrays supports the hypothesis that trimeric complexes of such annexins, including annexin IV, may represent the functional units that interact with membranes.