bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary function changes and immunomodulation of cytokine expression by zafirlukast after sensitization and allergen challenge in brown Norway rats
Authors: Lin C.‐C. , Lin C.‐Y. , Liaw S.‐F. , Chen A.
Published: March 2003

Background The cysteinyl leukotrienes are known important mediators in bronchial asthma.

Objective Our purpose was to evaluate the effect of zafirlukast on the late‐phase reaction, bronchial hyper‐responsiveness (BHR) and T cell‐related cytokine mRNA expression in ovalbumin (OA)‐sensitized brown Norway rats (BNRs).

Methods Thirty BNRs were equally divided into three groups. Group I and II animals were sensitized and then provoked with OA. Zafirlukast was given intraperitoneally (i.p.) to group I  animals prior to provocation. Group II animals received i.p. normal saline. Group III animals (controls) were not sensitized and breathed aerosolized saline. After OA provocation, the animals were anaesthetized. Pulmonary function tests (PFT) were performed at baseline and after varying doses of acetylcholine. Thereafter, bronchoalveolar lavage (BAL) was performed and the lungs were examined histologically. Total RNA was extracted from lung tissue and reverse transcriptase‐polymerase chain reaction (RT‐PCR) was performed using primers for IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, TNF‐α, IFN‐γ, iNOS and β‐actin.

Results Group II OA‐treated BNRs had worse PFT results, more severe bronchoconstriction in response to acetylcholine, and more severe inflammation in lung tissue than the other two groups. Group II had higher IL‐2, IL‐4, IL‐10 and IFN‐γ cytokine levels in BAL fluid and higher IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, IFN‐γ, TNF‐α and iNOS mRNA levels when compared with group I.

Conclusion Zafirlukast is effective in preventing late‐phase bronchoconstriction and BHR, reducing inflammatory response, and decreasing IL‐2, IL‐4, IL‐5, IL‐6, IL‐10 and IFN‐γ and iNOS mRNA expression.
Cysteinyl leukotrienes induce human eosinophil locomotion and adhesion molecule expression via a CysLT1 receptor‐mediated mechanism
Authors: Fregonese L. , Silvestri M. , Sabatini F. , Rossi G. A.
Published: March 2003

Background The mechanisms involved in eosinophil recruitment by cysteinyl‐leukotrienes (CysLTs) remain to be defined.

Objective We investigated whether CysLTs LTC4, LTD4 and LTE4 could directly stimulate in vitro adhesion molecule expression and cell locomotion of blood eosinophils from atopic asthmatic donors.

Methods Mab staining and FACS analysis were used to evaluate Mac‐1 and LFA‐1 expression on eosinophils before and after CysLTs stimulation. Eosinophil locomotion was tested using a 48‐well Boyden microchamber.

Results CysLTs, at the concentrations of 1 and 10 nM, were able to significantly up‐regulate Mac‐1 expression (P < 0.05, each comparison) but not LFA‐1 expression (P > 0.05, each comparison). A dose‐dependent, eosinophil chemotaxis was also induced by LTC4, LTD4 and LTE4 (0.1–10 nM) (P < 0.01, each comparison). Montelukast (0.01 nM to 10 nM), a specific CysLT1 receptor antagonist, significantly down‐regulated LTC4, LTD4 and LTE4‐induced Mac‐1 expression (P < 0.01, each comparison) and the CysLT‐induced eosinophil migration (P < 0.01, each comparison). In contrast, montelukast did not affect Mac‐1 expression or cell migration when eosinophils were stimulated by the ‘non‐specific activators’, such as fMLP or C5a (P > 0.05, each comparison).

Conclusion These data demonstrate that CysLTs are active in vitro in directly up‐regulating human eosinophil functions involved in eosinophil recruitment. The down‐regulation of Mac‐1 expression and eosinophil chemotaxis by the potent and selective CysLT1 receptor antagonist montelukast indicated the specificity of the LTC4‐, LTD4‐ and LTE4‐induced response.
Outcome of a latex avoidance program in a high‐risk population for latex allergy – a five‐year follow‐up study
Authors: Reider N. , Kretz B. , Menardi G. , Ulmer H. , Fritsch P.
Published: March 2003

Background Children with a shunted hydrocephalus are at highest risk for developing an immediate type allergy to latex. Limited data are available for preventive or therapeutical approaches.

Objective To evaluate the effectiveness of latex avoidance, with special regard to status of sensitization and compliance.

Methods In 1995, 131 children with a shunted hydrocephalus were screened for sensitization to latex by skin prick test and determination of specific IgE. Patients and parents were instructed on latex‐avoiding strategies. Hospital physicians, family doctors and dentists were advised to perform further surgical and other medical interventions under latex‐free conditions.

In 2000, 100 of these 131 patients were re‐evaluated according to the same testing procedures. Special attention was directed at the extent prophylaxis had been performed.

Results In 1995, 30/100 patients re‐evaluable in 2000 proved sensitized to latex, 70 had negative testing results. In 2000, 64/70 patients were still negative, six had meanwhile developed latex‐specific IgE. Seven out of thirty subjects with former positive testing had changes within the same RAST‐class, 20 showed a decline of at least one RAST‐class, whereas in three cases an increase of latex‐specific IgE was found. However, only 34 patients, mainly those being already sensitized, had thoroughly followed both medical and private prophylaxis. Within this group, 16 subjects (47.1%) had improved and another nine (26.5%) were still negative. Only three (8.8%) already previously sensitized patients presented with a further increase of latex‐specific IgE. Medical prevention contributed more to the outcome than home prevention. No statistically significant correlation with latex‐avoidance was observed, however, in previously unsensitized subjects. Underlying disease, atopy, number of operations, and age did not prove as significant variables.

Conclusion Secondary prevention results in a decrease of specific IgE in latex‐sensitized patients with hydrocephalus. This is due to medical more than home prophylaxis. Sensitization obviously occurs mainly in early childhood, thus primary prevention remains to be the main target.
Fluticasone inhibits the progression of allergen‐induced structural airway changes
Authors: Vanacker N. J. , Palmans E. , Pauwels R. A. , Kips J. C.
Published: March 2003

Background Inhaled corticosteroids are widely used as first‐line therapy in patients with asthma. The concept of early introduction is more and more accepted.

Objective In our rat model of airway remodelling, we investigated whether treatment with inhaled fluticasone propionate can inhibit further progression of established structural airway changes.

Methods Sensitized Brown Norway rats were exposed to aerosolized ovalbumin (1%) from day 14 to 42. From day 28 to 42, animals were treated with inhaled fluticasone or placebo 30 min before each allergen challenge. One control group was exposed to PBS from day 28 to 42, a second control group throughout the whole experiment.

Results Exposure to ovalbumin during 2 weeks induced structural airway changes, including epithelial cell proliferation, increase in airway wall area and fibronectin deposition. Goblet cell number was increased, although not significantly compared with PBS. Continuing allergen exposure for 2 weeks further enhanced each of these features. In addition, the amount of collagen in the airway wall was enhanced by 4 weeks allergen exposure compared with PBS‐exposed animals. These additional increases were inhibited by treatment with fluticasone during the last 2 weeks.

Conclusion The progression of established allergen‐induced structural airway changes in sensitized rats can be inhibited by treatment with fluticasone.
A double‐blind, placebo‐controlled study on the diagnostic accuracy of an electrodermal test in allergic subjects
Authors: Semizzi M. , Senna G. , Crivellaro M. , Rapacioli G. , Passalacqua G. , Canonica W. G. , Bellavite P.
Published: March 2003

Background Many unconventional diagnostic procedures based on bioelectrical skin responses are presently widely used for allergic diseases, but rigorous experimental evaluations of their accuracy are still lacking.

Aim We assessed whether an electrodermal device can correctly diagnose respiratory allergy.

Methods The diagnostic accuracy of the electrodermal device was assessed in double‐blind fashion in 72 allergic patients and 28 healthy volunteers. A random sequence of substances in sealed vials, including histamine, allergens, immunoglobulins at various dilutions and physiological saline, were tested in duplicate in each subject.

Results A wide variability of the measurements was found in most patients irrespective of their allergy status and of the substance tested. Allergic patients showed more negative skin electrical response at the second trial, compared to normal controls, independent of the tested substance. No significant difference in skin electrical response between allergens and negative controls could be detected.

Conclusion We conclude that the studied bioelectrical method, under blind testing, cannot correctly detect respiratory allergy.
Der p 2 isoallergens have different allergenicity, and quantification with 2‐site ELISA using monoclonal antibodies is influenced by the isoallergens
Authors: Park J. W. , Kim K. S. , Jin H. S. , Kim C. W. , Kang D. B. , Choi S. Y. , Yong T.‐S. , Oh S. H. , Hong C.‐S.
Published: March 2003

Background Der p 2 isoallergens have been reported and the possibility of different allergenicity has also been suggested. In addition, the quantification with 2‐site ELISA may be affected by the isoallergens.

Objectives Two different recombinant Der p 2 (rDer p 2) isoallergens were compared in terms of human IgE responses and the reliability of quantification of them with two‐site ELISA kits which use monoclonal antibodies (mAbs) as capture and detection of Der p 2.

Methods Seven different Der p 2 cDNA from the cultured Dermatophagoides pteronyssinus (DP) were cloned and polymorphism in nine amino acid residues was found. Two different recombinant isoallergens (rDer p 2A and rDer p 2B) were expressed and compared to their human IgE immune responses by ELISA and the ELISA inhibition test with 23 sera of DP‐allergic patients. The reliability of quantification of two different available 2‐site ELISA kits, which used mAbs for capture and detection of Der p 2, was evaluated.

Results The ELISA optical density of rDer p 2B‐specific IgE (sIgE) was higher than that of rDer p 2A (P < 0.001). The ELISA inhibition curve of rDer p 2B sIgE in pool I sera (n = 5; high sIgE both to rDer p 2A and rDer p 2B) did not show any differences in the 50% inhibition concentration and maximum inhibitory percentage of rDer p 2A and rDer p 2B sIgE. However, with pool II sera (n = 5; markedly higher sIgE to rDer p 2B than rDer p 2A), the 50% inhibitory concentrations (10 µg/mL vs. 40 ng/mL) and maximum inhibitory percentage (61% vs. 99%) of rDer p 2B sIgE with the two recombinant isoallergens were quite different. rDer p 2B could be quantified with two different 2‐site ELISA kits, but rDer p 2A was detected by only one kit.

Conclusion We conclude that isoallergens of Der p 2 may have different IgE immune responses. Quantification of Der p 2 with 2‐site ELISA kits that adopted mAbs, might be affected by the prevalent form of the isoallergens in reservoir dust.
Eosinophilic inflammation and airway hyper‐responsiveness are profoundly inhibited by a helminth (Ascaris suum) extract in a murine model of asthma
Authors: Lima C. , Perini A. , Garcia M. L. B. , Martins M. A. , Teixeira M. M. , Macedo M. S.
Published: March 2003

Background The increase of atopic disorders in developed countries has been associated with the decline of infectious diseases, including helminthic infections. We have already demonstrated that adult worm extracts from Ascaris suum (ASC) suppress the IgE antibody production against unrelated antigens.

Objective Here we investigated the influence of ASC on the development of pulmonary eosinophilic inflammation in a murine model of asthma.

Methods Heat‐coagulated egg white alone (EWI) or mixed with ASC (EWI + ASC) was implanted subcutaneously in B10.A or C57BL/6 mice, and 14 days later they were challenged intratracheally with OVA or exposed to aerosolized OVA for 4 days.

Results The suppressive effect of ASC was demonstrated on the accumulation of cells into airways, with reduction of eosinophil numbers and of eosinophil peroxidase activity in EWI + ASC‐ immunized mice. This effect correlated with a marked reduction of IL‐5 and IL‐4 levels in the BAL from C57BL/6 and B10.A mice, respectively, and of eotaxin in BAL and lung tissue from both strains. OVA‐specific IgG1 and IgE levels were also impaired in serum and BAL from these mice. Airway hyper‐reactivity to methacholine was obtained in B10.A mice sensitized with EWI, but the respiratory mechanical parameters returned to normal levels in EWI + ASC‐immunized mice.

Conclusion These results indicate that ASC has a profound inhibitory effect on lung inflammation and hyper‐responsiveness and that suppression of IL‐5 or IL‐4 and of eotaxin contributes to this effect.
Induction of allergic inflammation in the lungs of sensitized sheep after local challenge with house dust mite
Authors: Bischof R. J. , Snibson K. , Shaw R. , Meeusen E. N. T
Published: March 2003

Background Previous sheep models of asthma are based on sheep sensitized to nematode (Ascaris) allergens and these have been used to evaluate the physiological and pharmacological effects of potential anti‐asthma agents. The immunological mechanisms associated with the allergic response in sheep lungs has not been examined in detail.

Objective To develop an experimental sheep model of allergic lung inflammation based on a relevant major human allergen, house dust mite, and to define the immunological features of the allergic response in this model.

Methods Sheep immunized subcutaneously with solubilized house dust mite extract were given a single bronchial challenge with house dust mite. Bronchoalveolar lavage (BAL) and peripheral blood leucocytes were collected before and after challenge for flow cytometry, and tissue samples were taken post‐mortem (48 h post‐challenge) for histology and immunohistochemical analyses.

Results Immunizations with 50 μg house dust mite induced an allergen‐specific IgE response in 50 to 60% of sheep (allergic sheep), with higher antigen doses increasing specific IgG1 but not IgE. Lung challenge of allergic sheep with house dust mite led to the initial recruitment of neutrophils (at 6 h post‐challenge) followed by eosinophils and activated lymphocytes into the lung tissue and BAL, similar to the late‐phase allergic response seen in human asthma. Eosinophil recruitment peaked at 48 h post‐challenge, representing 10 to 33% of BAL leucocytes in allergen‐challenged allergic sheep compared to 0 to 3% in allergen‐challenged control (naïve) sheep. Lymphocytes recovered from the lung after allergen challenge were enriched for CD4+ T cells and were more activated than lymphocytes in blood. There was significant down‐regulation of CD62L (L‐selectin) and CD49d (VLA‐4) expression after allergen challenge on BAL eosinophils and lymphocytes compared to blood. In addition, VCAM‐1 (ligand for VLA‐4) was up‐regulated on blood vessels of allergen‐challenged lungs. Eosinophils, CD4+ T cells and CD45R+ B cells were the most prominent leucocytes found in lung tissue 48 h after allergen challenge.

Conclusion This study demonstrates, for the first time, the ability of house dust mite to induce allergic responses in sheep lungs. This novel sheep model of allergic lung inflammation using relevant human allergens, exhibits similarities to human asthmatic disease and will be a useful tool for studies of the immunological and physiological mechanisms of allergic asthma.
Hymenoptera ultra‐rush venom immunotherapy (210 min): a safety study and risk factors
Authors: Birnbaum J. , Ramadour M. , Magnan A. , Vervloet D.
Published: March 2003

Background In this study, which summarizes our last 5 years of experience, we evaluated the side‐effects of ultra‐rush venom immunotherapy and the possibility to define some risk factors for side‐effects as age, Hymenoptera venom used for treatment, treatment phase, severity of prior insect sting reaction, concentration of skin test positivity, and level of specific IgE.

Methods In our protocol on day 1, an initial venom dose of 0.1 μg was followed by 1, 10, 20 μg at 30‐min intervals and then 30 and 40 μg at 60‐min intervals. Patients who completed this protocol received two booster injections of 50 μg on day 15 and one of 100 μg on day 45. Subsequently, monthly 100 μg boosters were given.

Results Fifty‐one children (9.20 ± 3.41 years) and 207 adults (40.62 ± 14.00 years) underwent an ultra‐rapid venom immunotherapy (ultra‐RVIT). Single ultra‐RVIT was administered to 195 patients: 69 with honeybee, 123 with yellow jacket and three with wasp venoms. Two venoms were injected into 59 patients: 42 with yellow jacket and wasp, 17 with yellow jacket and honeybee. Four patients received the three venoms.

The frequency of immediate systemic reactions (SR) was not significantly different between adults and children: 11.2% vs. 10.8%.

SR were experienced more frequently on day 1 (n= 33). They were uncommon on day 15 (n= 2) and on day 45 (n= 1). No late reactions have been observed. Honeybee venom induced significantly more SR (30%) vs. yellow jacket (3.2%) and wasp venom (6.1%). Among the 33 patients who experienced a SR on day 1, 24 had a reaction grade 1 or 2 and nine had a reaction grade 3 or 4. There is a significant risk for patients with a prior reaction grade 3 or 4 to experience a SR during venom immunotherapy (VIT).

The strength of positive skin tests and the level of specific IgE were not related to an increased risk of SR (P= NS).

Conclusion Treatment with honeybee extract induced more SR than the treatment with yellow jacket and wasp venom. Doses increase phase on day 1 is risk factors for SR of ultra‐RVIT, as well as the severity of the prior reaction. Age, degree of positive skin tests, and specific IgE are not risk factors.
Mouse serum amyloid A (SAA) proteins isolated by two-dimensional electrophoresis: characterization of isotypes and the effect of separate and combined administrations of cytokines, dexamethasone and lipopolysaccharide (LPS) on serum levels and isotype distribution
Authors: Foyn Bruun C , Sletten K , Marhaug G
Published: March 2003

Hydrophobic interaction chromatography and two-dimensional electrophoresis were used to isolate and characterize mouse SAA, and to study the in vivo effect of separate or combined administrations of cytokines, dexamethasone (DEX) and LPS on mouse SAA. Four SAA spots containing partial amino acid sequence in accordance with mouse apoSAA1 and apoSAA2/SAASJL/J pI 5.9 were demonstrated in serum. One of these proteins represents a previously undescribed, acidic acute-phase mouse SAA protein. Both DEX and interferon-gamma (IFN-γ) proved to be capable of increasing SAA serum levels. In contrast to what has been shown in previous in vivo studies, administration of IL-6 did increase the SAA levels to nearly the same magnitude as IL-1, and the effect of IL-6 and LPS on SAA production was not significantly altered by the addition of DEX. Irrespective of the inflammatory stimuli that was administered, a non-selective production of SAA1 and SAA2 was observed in most groups, including the group that received IL-6. The results illustrate that data obtained about mouse SAA are highly dependent on which models, isolation and identification methods are used.
Abnormal intracellular IL-2 and interferon-gamma (IFN-γ) production as HIV-1-associated markers of immune dysfunction
Authors: Westby M , Marriott J B , Guckian M , Cookson S , Hay P , Dalgleish A G
Published: March 2003

We used three-colour cytometry to analyse intracellular cytokine production in activated whole blood cultures derived from patients with HIV-1 infection. We assessed mitogen-induced IL-2, IL-4 and IFN-γ production from T cells as possible markers of immune dysfunction. The percentages of T cells staining for IL-2 were significantly reduced in stimulated cultures from HIV+ individuals relative to normal controls (P < 0.0001); this reduction was observed in both the CD4+ and the CD8+ subsets. IL-2 production was significantly reduced in CD4+ T cells from HIV+ individuals clinically classified as symptomatics compared with HIV+ asymptomatics (P < 0.001); in addition, production of IL-2 inversely correlated with viral load (r2 = 0.832). On the other hand, HIV+ individuals showed significantly more T cells staining positive for IFN-γ (P < 0.0001); subset analysis identified these T cells as CD8+. Increased IFN-γ production in the CD8+ T cell subset of HIV+ individuals correlated neither with clinical status nor with plasma viral load. IL-4 staining in activated T cells was low (< 5%) and no differences were observed between HIV+ and control groups. Three-colour FACS analysis of whole blood provides a sensitive, rapid and relatively easy means to detect cytokine profiles within T cell subpopulations. Only small volumes of blood are required (0.5 ml), since there is no need for cell isolation, making it more practical than ELISA or reverse transcriptase-polymerase chain reaction (RT-PCR) for the analysis of immune function in HIV+ individuals. This technique could therefore play a role in mapping the dynamics and extent of immune recovery in AIDS patients undergoing triple combination therapy.
Anti-mitochondrial M5 type antibody represents one of the serological markers for anti-phospholipid syndrome distinct from anti-cardiolipin and anti-β2-glycoprotein I antibodies
Authors: La Rosa L , Covini G , Galperin C , Catelli L , Del Papa N , Reina G , Morabito A , Balestrieri G , Tincani A , Gershwin M E , Meroni P L
Published: March 2003

The aim of this study was to characterize the antigen specificity and to evaluate the diagnostic and prognostic value of anti-mitochondrial M5 type antibodies (AMA M5). Fifty-eight patients selected on the basis of their AMA M5 positivity were investigated in relationship to their clinical and serological profile. Cross-absorption studies, Western blotting and immunoprecipitation analysis were carried out for AMA M5 antigen specificity characterization. Most patients had a diagnosis of systemic lupus erythematosus (SLE) (65.5%) or of primary anti-phospholipid syndrome (PAPS) (24%); all the patients were positive for IgG or IgM anti-cardiolipin (anti-CL) antibodies and 49% of them also displayed lupus anticoagulant (LA) activity. Anti-β2-glycoprotein I (β2-GPI) IgG were detectable in 30/38 sera (78.9%) and IgM in 34/38 (89.4%). While anti-CL and anti-β2-GPI IgG antibodies were significantly associated with history of thrombosis and fetal loss, AMA M5 displayed a statistical association only for thrombocytopenia and recurrent fetal loss. Absorption with human β2-GPI both in free solution or in solid phase as well as with CL liposomes or CL/β2-GPI liposome complexes did not affect AMA M5 fluorescence. While AMA M5 activity is absorbed by whole mitochondrial preparations, no specific reactivities against several human, bovine and rat mitochondrial proteins could be detected in Western blotting and immunoprecipitation studies. AMA M5 appear to be detectable in both primary and secondary APS, displaying a strong association with the presence of thrombocytopenia and fetal loss. Although strictly related to anti-phospholipid antibodies, AMA M5, anti-CL and anti-β2-GPI antibodies represent distinct serological markers of the APS.
Genetic identification of antigens exposed in damaged endothelial cells as laminin-binding proteins
Authors: Ireland D C , Spring E L , Moiseeva E , DE Bono D P
Published: March 2003

A monoclonal antibody, D5G2, which reacts in a balloon angioplasty damage model with unfixed damaged but not with unfixed undamaged human endothelial cells, was used to screen a human endothelial cDNA library in an Escherichia coli/λ gt11 expression system. Sequences of DNA inserts in D5G2+ phage clones matched those reported for a laminin-binding protein, LBP-32. Both D5G2 and purified laminin bound to a polypeptide of 55 kD on PVDF membranes carrying electrophoretically separated endothelial cell lysates, D5G2 also bound to recombinant LBP expressed in E. coli, and showed similar staining patterns on human and bovine endothelial cells to another characterized anti-LBP antibody. Increased staining of unfixed endothelial cells on detergent permeabilization suggests that D5G2 binds to intracellular laminin-binding protein made accessible by cell membrane injury. Antibodies to intracellular targets exposed by cell damage may be useful in anchoring therapeutic agents at sites of vascular damage.
VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)
Authors: Huang S-C , Jiang R , Hufnagle W O , Furst D E , Wilske K R , Milner E C B
Published: March 2003

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of VH genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of VH3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged VH4 genes was also observed in these patients. These data suggest that although usage of individual VH genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.
Recombinant α-chains of type IV collagen demonstrate that the amino terminal of the Goodpasture autoantigen is crucial for antibody recognition
Authors: Ryan J J , Mason P J , Pusey C D , Turner N
Published: March 2003

Goodpasture's disease, an autoimmune disorder causing severe glomerulonephritis and pulmonary haemorrhage, is characterized by antibodies to the glomerular basement membrane (GBM). The principal target antigen has been identified as the carboxyl terminal non-collagenous (NC1) domain of the α3-chain of type IV collagen. Anti-GBM antibodies appear to recognize one major epitope that is common to all patients, and is largely conformational. We have analysed antibody binding to recombinant α(IV)NC1 domains using a construct and expression system shown to produce correctly folded antigen that is strongly recognized by autoantibodies. In this system, as with the native antigen, α3(IV)NC1 was bound strongly by antibodies from all patients, whereas the closely related α1(IV) and α5(IV)NC1 domains, similarly expressed, showed no such binding. A series of chimeric NC1 domains, between human α3(IV) and α1(IV), and between human and rat α3(IV), were expressed as recombinant molecules, and were recognized by autoantibodies to varying degrees. Strong binding required the presence of human α3(IV) sequence in the amino terminal region of both sets of chimeric molecules. This work strongly suggests that the amino terminal of α3(IV)NC1 is critical for antibody recognition, whereas the carboxyl terminal end of α3(IV)NC1 has a less important role.
Fas/Fas ligand (FasL)-deregulated apoptosis and IL-6 insensitivity in highly malignant myeloma cells
Authors: Frassanito M A , Silvestris F , Silvestris N , Cafforio P , Camarda G , Iodice G , Dammacco F
Published: March 2003

IL-6 is a growth factor which interferes in the apoptosis of malignant plasma cells. Here we explore its role in the spontaneous and Fas/FasL-regulated apoptosis of seven myeloma cell clones (MCC). MCC-2 and -7 were constitutively defective in Fas antigen in the presence of large membrane exposure of FasL, and showed a high rate of cell proliferation irrespective of the presence of IL-6. Cytofluorimetric analysis following propidium iodide (PI) staining revealed a minimal extent of spontaneous apoptosis, as in other IL-6-insensitive, though Fas-positive MCC, namely MCC-3 and -5. By contrast, a regular amplitude of apoptosis occurred in the remaining IL-6-dependent clones. Their propensity to cell death, as well as their FasL membrane expression, were promptly down-modulated by the cytokine, whereas no substantial effect was detected in IL-6-independent MCC. Furthermore, we investigated the quantitative secretion of FasL. Both [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide] (MTT) cytotoxicity assay and PI staining of WC8 lymphoblasts from a Fas-transfected mouse lymphoma, incubated with supernatants from MCC, showed a variable cytocidal property, thus confirming the cellular release of FasL. However, a significant elevation of FasL secretion occurred in both Fas− MCC, whereas molecular cloning and sequencing of Fas revealed the presence of a splicing variant, namely Fas Exo4,6Del, in the cDNA from both MCC-3 and -5, which were previously demonstrated to be unresponsive to Fas stimulation. Taken together, these data provide evidence that concurrence of IL-6 insensitivity and deregulation of apoptosis in myeloma cells reflects a high malignancy grade. It is suggested that the secretion of Fas splicing variants in Fas+ plasma cells, as well as the over-production of FasL in Fas− myelomas, are differential mechanisms by which myeloma cells escape host immune surveillance.
Human intestinal epithelial cells down-regulate IL-8 expression in human intestinal microvascular endothelial cells; role of transforming growth factor-beta 1 (TGF-β1)
Authors: Lügering N , Kucharzik T , Gockel H , Sorg C , Stoll R , Domschke W
Published: March 2003

Cytokines produced from intestinal epithelial cells may function as signals to neighbouring immune cells. In the present study we analysed the effects of colonic epithelial cell lines (HT-29, Caco-2, HCT-116, Colo-320) and freshly isolated intestinal epithelial cells on IL-8 expression in the SV-40T transfected human microvascular endothelial cell line (HMEC-1). Epithelial cell-conditioned media and transwells preventing physical contact between epithelial and endothelial cells were used. TGF-β1 and IL-8 levels were determined by ELISA and Northern blot analysis. Increasing concentrations of IL-1β led to increasing production of IL-8. The addition of epithelial cell-conditioned medium or epithelial cells to HMEC-1 cells in a two-compartment co-culture system resulted in a strong decrease in IL-8 at the protein and mRNA level. Decrease of IL-8 was markedly stronger when epithelial cells were co-cultured in contact with HMEC-1 cells, indicating that not only soluble factor(s) play a role in the induction of IL-8 suppression in HMEC-1 cells. MoAbs against TGF-β1 partially inhibited down-regulation of endothelial IL-8 expression. In further studies, IL-8 expression in freshly isolated human intestinal microvascular endothelial cells (HIMEC) was also down-regulated by intestinal epithelial cells. Our results demonstrate that intestinal epithelial cells down-regulate IL-8 expression in HMEC-1 cells. TGF-β1 is a candidate factor of epithelial–endothelial communication in the colonic mucosa.
Antibody to carbonic anhydrase II is present in primary biliary cirrhosis (PBC) irrespective of antimitochondrial antibody status
Authors: Invernizzi P , Battezzati P M , Crosignani A , Zermiani P , Bignotto M , Del Papa N , Zuin M , Podda M
Published: March 2003

Antibody to carbonic anhydrase II, an enzyme abundantly present in biliary epithelium, has been proposed as a diagnostic marker for antimitochondrial antibody-negative PBC. In this study we determine its prevalence and clinical significance in a large series of patients with antimitochondrial antibody-positive and -negative PBC. Reactivity to carbonic anhydrase II was sought by Western immunoblotting in sera from 215 consecutive patients with PBC (26 antimitochondrial antibody-negative), 13 with autoimmune hepatitis, 25 with primary Sjögren's syndrome (pSS), 12 with systemic sclerosis, 19 with systemic lupus erythematosus and 73 healthy subjects. The prevalence of antibody to carbonic anhydrase II (titre 1:100) in PBC was 8%. No specific reactivity to carbonic anhydrase II was found in antimitochondrial antibody-negative PBC (7% versus 8% in antimitochondrial antibody-positive PBC). Ascites (P = 0.006) and Sjögren's syndrome (SS) (P = 0.022) in PBC were significantly associated with presence of the antibody. In patients with SS associated with PBC, the prevalence (19%) was similar to that observed in pSS (16%). At a serum dilution of 1:40, the prevalence of positive sera in PBC rose to 27% but disease specificity was reduced. Our findings in a large population of PBC patients rule out a relation between presence of antibody to carbonic anhydrase II and lack of antimitochondrial antibody. The higher prevalence of ascites found in positive patients warrants further evaluation.
Circulating cytokine balance and activation markers of leucocytes in Q fever
Authors: CAPO C , AMIRAYAN N , GHIGO E , RAOULT D , MEGE J-L
Published: March 2003

As Q fever is associated with an inflammatory syndrome, we determined circulating levels of inflammatory cytokines, cytokine antagonists, and activation markers of leucocytes in patients with acute Q fever and Q fever endocarditis. Tumour necrosis factor (TNF) and IL-6, but not IL-1β, were markedly increased compared with controls. Cytokine antagonists and activation markers of leucocytes were profoundly different in acute and chronic Q fever. IL-1 receptor antagonist and TNF receptor type II were significantly increased in patients with acute Q fever, suggesting a shift of cytokine balance towards cytokine antagonists. The activation marker of B cells, sCD23, was significantly increased in Q fever endocarditis compared with controls and patients with acute Q fever. In a 2-year follow-up study of patients with Q fever endocarditis, sCD23 and specific IgG levels slowly decreased in patients whose symptoms resolved, but remained high in those who required prolonged treatment.
Idiotype usage by polyclonally activated B cells in experimental autoimmunity and infection
Authors: SUTMULLER M , BAELDE J J , MADAIO M P , BRUIJN J A , DE HEER E
Published: March 2003

Both in animal models and in human systemic lupus erythematosus (SLE) the occurrence of nephritogenic autoantibodies bearing dominant idiotypes has been described. In this study we investigate the relation between the induction pathway of polyclonal B cell activation and the production and glomerular deposition of nephritogenic antibodies with shared dominant idiotype(s). Polyclonal B cell activation was induced in several experimental models characterized by glomerular immune deposit formation. We monitored the occurrence of dominant idiotypes among immunoglobulins deposited in the glomeruli. In addition, we studied the species specificity of the dominant idiotypes, by monitoring their presence in kidney sections of patients with an immunologically mediated kidney disease. Anti-idiotype antisera against two monoclonal anti-DNA autoantibodies were used, derived from MRL-lpr/lpr mice, i.e. clone H241 and clone H130. Autoantibodies with the H241 idiotype were present in immune complex depositions in all experimental models but not in humans. We therefore conclude that the presence of this dominant idiotype is independent of the induction pathway of polyclonal B cell activation. However, autoantibodies bearing the H130 idiotype were only detected in kidney sections of mice with spontaneous lupus.
T cell responses to the putative dominant autoepitope in primary biliary cirrhosis (PBC)
Authors: PALMER J M , DIAMOND A G , YEAMAN S J , BASSENDINE M F , JONES D E J
Published: March 2003

PBC is characterized by T cell-mediated destruction of the biliary epithelial cells lining the small intrahepatic bile ducts. The E2 and E3 binding protein (E3BP (protein X)) components of pyruvate dehydrogenase complex (PDC) are disease-specific autoantigens in PBC. Attempts to localize the T cell autoepitopes within PDC-E2 have, however, generated contradictory results. One study has suggested the presence of T cell epitopes throughout PDC-E2, whilst another has identified a single dominant 14 amino acid T cell epitope (p163) spanning the lipoic acid binding lysine residue in the inner lipoyl domain (ILD) of PDC-E2. The aim of the current study was to determine the prevalence of T cell responses to p163 and PDC-E2 ILD, and the role played by lipoylation of these antigens in their immunogenicity, in a UK PBC population. We found that the majority of the PBC patients showing a 6-day peripheral blood T cell proliferative response to native human PDC also responded, in a MHC class II-restricted fashion, to biochemically purified PDC-E2 and E3BP (which co-purify) (9/10 positive (SI > 2.76), mean SI 5.74 ± 5.04 (PDC-E2/E3BP) versus 6.67 ± 3.84 (PDC), P = NS), implying that the important PBC-specific T cell epitopes are contained within the PDC-E2 or E3BP components of PDC. Only a minority of patients responsive to PDC, however, responded to either lipoylated recombinant PDC-E2 ILD (4/10 positive, mean SI 1.98 ± 1.24, P < 0.005 versus PDC response) or lipoylated p163 (4/12 positive, mean SI 1.90 ± 1.58, P < 0.001). The lipoylation state did not affect the T cell response to either ILD or p163. Our findings suggest that in some UK patients with PBC there are immunodominant T cell autoepitopes within PDC-E2/E3BP which are outside the ILD of PDC-E2.
Glucocorticoids modulate the development of dendritic cells from blood precursors
Authors: VAN DEN HEUVEL M M , VAN BEEK N M A , BROUG-HOLUB E , POSTMUS P E , HOEFSMIT E C M , BEELEN R H J , KRAAL G
Published: March 2003

Dendritic cells (DC) are professional antigen-presenting cells, capable of priming naive T cell responses. Glucocorticoids (GC) are frequently used in asthmatic patients. In this study we describe the effects of GC on the development and function of monocyte-derived DC (MoDC) in vitro and in vivo. Monocytes from healthy individuals were isolated and incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days, to induce maturation into MoDC. To study the role of GC on DC differentiation in vitro cells were incubated with dexamethasone at different stages of MoDC development. At day 6 cells were characterized phenotypically by flow cytometry and functionally in an allogeneic mixed leucocyte reaction. To study the effect of GC in vivo patients with mild/moderate atopic asthma were selected. In one group no GC were used, whereas the other group used inhalation GC. MoDC from these patients were generated as described above and tested functionally. Incubation of MoDC or its peripheral blood precursors with dexamethasone decreased the accessory potency dose-dependently. The functional differences could not be explained by the changes in the expression of MHC II and the costimulatory molecules CD40 and CD86. The relevance of this mechanism was confirmed for the in vivo situation as well. MoDC from patients using inhalation GC showed a decreased accessory potency. These data suggest a modulatory effect of GC therapy at the level of the peripheral blood monocyte. The results indicate that GC influence DC development and function in vitro as well as in vivo.
Rapid intestinal ischaemia-reperfusion injury is suppressed in genetically mast cell-deficient Ws/Ws rats
Authors: ANDOH A , KIMURA T , FUKUDA M , ARAKI Y , FUJIYAMA Y , BAMBA T
Published: March 2003

Ws/Ws rats have a small deletion of the c-kit gene, and are deficient in both mucosal and connective tissue-type mast cells. In this study, the role of mucosal type mast cells (MMC) in the development of intestinal ischaemia-reperfusion injury was investigated in Ws/Ws rats. Autoperfused segments of the jejunum were exposed to 60 min of ischaemia, followed by reperfusion for various time periods. The epithelial permeability was then assessed by the 51Cr-EDTA clearance rate. In the control (+/+) rats, the maximal increase in mucosal permeability was achieved at 45 min of reperfusion. In contrast, this increase was significantly and potently attenuated in the Ws/Ws rats. Mucosal alkaline phosphatase activity decreased in the control (+/+) rats, but was not altered in the Ws/Ws rats. There were no differences in mucosal myeloperoxidase activity, indicating that granulocytes did not contribute to tissue injury. These results provide direct evidence for the role of mast cells in the pathogenesis of intestinal ischaemia-reperfusion injury.
Surface phenotype analysis of CD16+ monocytes from leukapheresis collections for peripheral blood progenitors
Authors: TANAKA M , HONDA J , IMAMURA Y , SHIRAISHI K , TANAKA K , OIZUMI K
Published: March 2003

In peripheral blood progenitor cell (PBPC) collections from patients with solid tumour or haematological malignancy, monocytes were separated into two subpopulations. The majority of monocytes expressed CD14 at a high density without CD16 antigen (the CD14+CD16− monocytes). The remaining monocytes co-expressed CD14 and CD16 (the CD14+CD16+ monocytes). These CD14+CD16+ monocytes amounted to 20.6 ± 15.8%, while those in peripheral blood (PB) obtained from healthy volunteers were 7.3 ± 3.1% (P < 0.05). When subdividing the CD14+CD16+ monocytes into CD14brightCD16dim and CD14dimCD16bright cells, both populations were found to be increased in PBPC collections. Since typical CD14+CD16+ monocytes are the CD14dimCD16bright population, we compared the additional surface antigens on CD14dimCD16bright monocytes with those of CD14+CD16−monocytes. In PBPC collections, the CD14dimCD16bright monocytes exhibited lower levels of CD11b, CD15, CD33 and CD38 expression and higher levels of CD4, CD11a, CD11c and MHC class II, and also revealed a higher percentage of CD4+ cells and a lower percentage of CD15+ cells and CD38+ cells, compared with the CD14+CD16− monocytes. When compared with the CD14dimCD16bright monocytes in PB, those in PBPC collections exhibited higher expression of CD4 and lower expression of CD11b, and also showed higher percentages of CD4+ cells and CD38+ cells and a lower percentage of CD11b+ cells. These results suggest that PBPC collections may be rich in the CD14+CD16+ monocytes in which the proportion of the immature population is increased. It is likely that these monocytes participate in the haematological and immune recovery after PBPC transplantation.
Immunomodulatory effects ofLactobacillus plantarumcolonizing the intestine of gnotobiotic rats
Authors: HERÍAS M V , HESSLE C , TELEMO E , MIDTVEDT T , HANSON L Å , WOLD A E
Published: March 2003

We have studied the effect of the probiotic strain Lactobacillus plantarum 299v on the immune functions of gnotobiotic rats. One group of germ-free rats was colonized with the type 1-fimbriated Escherichia coli O6:K13:H1 and another group with the same E. coli strain together with L. plantarum 299v. One and 5 weeks after colonization, bacterial numbers were determined in the contents of the small intestine, caecum and mesenteric lymph nodes. Small intestinal sections were examined for CD8+, CD4+, CD25+ (IL-2R α-chain), IgA+ and MHC class II+ cells and mitogen-induced spleen cell proliferation was determined. Immunoglobulin levels and E. coli-specific antibodies were measured in serum. Rats given L. plantarum in addition to E. coli showed lower counts of E. coli in the small intestine and caecum 1 week after colonization compared with the group colonized with E. coli alone, but similar levels after 5 weeks. Rats colonized with L. plantarum+ E. coli had significantly higher total serum IgA levels and marginally higher IgM and IgA antibody levels against E. coli than those colonized with E. coli alone. They also showed a significantly increased density of CD25+ cells in the lamina propria and displayed a decreased proliferative spleen cell response after stimulation with concanavalin A or E. coli 1 week after colonization. The results indicate that L. plantarum colonization competes with E. coli for intestinal colonization and can influence intestinal and systemic immunity.
Dysregulation of B7.2 (CD86) expression on monocytes of HIV-infected individuals is associated with altered production of IL-2
Authors: KUMAR A , ANGEL J B , AUCOIN S , CREERY W D , DAFTARIAN M P , CAMERON D W , FILION L , DIAZ-MITOMA F
Published: March 2003

T helper (Th) responses are mediated in part by immunoregulatory cytokines and the signals delivered by the costimulatory CD28–B7 pathway. In this study, we have investigated the relationship between the regulation of B7 isoform expression on antigen-presenting cells from HIV+ individuals and the production of Th cytokines. The level of expression of both B7.1 and B7.2 isoforms as measured by mean channel fluorescence was significantly decreased on freshly isolated monocytes from HIV+ individuals compared with HIV− controls. However, the levels of expression of B7.1 and B7.2 on both B cells and monocytes increased significantly following culture in HIV+ individuals compared with HIV− controls. B7 expression is subject to regulation by immunoregulatory cytokines. Therefore, we analysed the regulation of B7 expression by cytokines, namely IL-10 and tumour necrosis factor-alpha (TNF-α), the production of which is enhanced in HIV infection and have similar inhibitory effects on B7 expression. Two groups of HIV+ individuals were distinguished on the basis of the inhibitory effect of IL-10 and TNF-α on monocyte B7.2 expression. IL-10 inhibited B7.2 expression on monocytes from some HIV+ individuals (termed responders) like the HIV− controls. However, in a subset of HIV+ individuals (non-responders) this inhibitory effect was lost. Loss of inhibition of B7.2 expression by IL-10 was associated with significantly reduced IL-2 production by phytohaemagglutinin (PHA)- stimulated peripheral blood mononuclear cells (PBMC). These observations showing an association of B7 dysregulation on monocytes and B cells with altered production of IL-2 may have implications in HIV immunopathogenesis.