bluuebunny/crossref_metadata_2025 huggingface dataset:
In situ hybridization analysis of ICAM‐1 (CD54) mRNA on conjunctival epithelium during allergic inflammation
Authors: BAGNASCO M. , PESCE G. , FIORINO N. , RICCIO A. M. , CIPRANDI G. , BUSCAGLIA S. , CANONICA G. W.
Published: November 2003

Background
The intercellular adhesion molecule ICAM‐1 has been detected by immunohistochemical methods on epithelial cells of the conjunctiva and nose during allergic inflammation.

Objective
The aim of the present study was to evaluate whether ICAM‐1 expression on conjunctival epithelium derives from endogenous synthesis or is merely due to passive uptake of soluble ICAM‐1 released from inflammatory cells.

Methods
In situ hybridization was performed using a 3’end dygoxygenin‐labelled specific DNA oligonucleotide probe on fixed conjunctival smears from allergic subjects challenged with, or naturally exposed to the allergen, and from healthy subjects. Immunocytochemistry for ICAM‐1 was performed by alkaline phosphatase antialkaline phosphatase.

Results
Results In allergic patients, both naturally exposed to the allergen and after specific challenge, a clear hybridization pattern on epithelial cells was apparent. Out of allergen exposure, some symptomfree pollinosic subjects, as well as a few healthy volunteers showed mild ICAM‐1 mRNA cytoplasmic staining in the absence of immunohistochemically detectable ICAM‐1. This finding may explain the very early appearance of ICAM‐1 on conjunctival epithelium following specific challenge in allergic individuals.

Conclusions
These results indicate that the presence of ICAM‐1 on conjunctival epithelium during allergic inflammation derives from endogenous synthesis and not from uptake of soluble ICAM‐l.
Specific induction of interleukin‐4‐producing cells in response to in vitro allergen stimulation in atopic individuals
Authors: GABRIELSSON S. , PAULIE S. , RAK S. , LAGGING E. , VAN HAGE‐HAMSTEN M. , HARFAST B. , TROYE‐BLOMBERG M.
Published: November 2003

Background and Objective
CD4+ T cells can be divided into two major subsets. T helper (TH)1 and TH2 cells. Interleukin‐4 (IL‐4) is produced by TH2 cells and induces switching of immunoglobulin (Ig) M/lgG to IgE. Interferon‐γ (IFNγ) produced by TH1 cells counteracts the IgE‐promoting effects of IL‐4. In this study we wanted to investigate whether the number of IL4‐producing cells could be a direct measurement of allergen exposure in vitro, and whether this was correlated to the elevated serum IgE‐levels seen in atopic persons.

Methods
We compared the number of IL‐4‐ and IFNγ‐producing cells using an enzyme‐linked immunospot assay (ELISPOT) in response to allergens from birch and cat in peripheral mononuclear cells from atopic and healthy individuals.

Results
In the two sensitized groups there was an increase in the number of lL‐4producing cells in response to the specific allergen which was not seen in the healthy group (1/20000 cells and 1/200 000 cells, respectively, P < 0.001 for birch). In criss‐cross experiments where birch‐sensitized individuals were stimulated with cat allergen, no lL‐4‐producing cells were seen, indicating a high degree of specificity. In individual subjects, the elevated numbers of IL‐4‐producing cells were significantly correlated with their allergen‐specific serum IgE levels. When allergen was combined with a suboptimal dose of PHA, there was a synergistic increase in the number of allergen‐induced IL‐4‐producing cells (1/4000 cells) in the atopic donors, which was not seen with the number of IFNγ‐producing cells.

Conclusions
Allergen‐specific IL‐4 producing cells in a peripheral blood mononuclear cell (PBMC) culture can be detected by ELISPOT and the response can synergistically be enhanced by suboptimal concentrations of PHA.
Reduction of exposure to laboratory animal allergens in the workplace
Authors: GORDON S. , WALLACE J. , COOK A. , TEE R. D. , TAYLOR A. J. NEWMAN
Published: November 2003

Objectives
Evidence is now accumulating that the prevalence of allergy to laboratory animals is related to the intensity of exposure to animal allergens. Whilst airborne animal allergen concentrations may be influenced by the litter type, cage design and stock density, the effectiveness of methods to reduce personal exposure has not been objectively assessed. Methods Air samples were collected at 2 L/min and 180L/min onto polytetrafluoroethylene (PTFE) filters and the rat urinary aeroallergen (RUA) and mouse urinary aeroallergen (MUA) concentrations were measured by radioallergosorbent test (RAST) inhibition.

Results
When 545 mice (11.1 mice/m3) were housed in ventilated cages (Thoren Maximiser cage system) operated at positive pressure to the environment, the static MUA concentration (n= 24. median = 0.10μg/m3) was reduced sevenfold when compared with conventional cage systems (n= 12, median = 0.67μ/g/m3, P < 0.001). MUA could be further reduced if the ventilated cage system was operated at lower pressure: static samples (n= 1) collected at 180 L/min at negative, ambient and positive pressure registered < 0.003, 0.02 and 0.28 μg/m3, respectively. During cleaning out, the intensity of personal exposure to RUA was apparently reduced twofold when soiled litter was removed by vacuum (n= 17, median = 22.87 μg/m3) when compared with tipping (n=18, median = 38.l.5μg/m3), P= 0.002) although the task took twice as long to perform. The RUA exposure associated with handling rats was reduced 25‐fold when performed in a ventilated cabinet (n= 21, median = 2.67 μg/m3) compared with handling of rats on an open bench (n= 17, medians 54.39 μg/m3, P= 0.0001).

Conclusions
Effective reduction of exposure to animal allergens can now be achieved by the use of ventilated systems both for housing and handling rats and mice providing safety equipment is used correctly. The vacuum removal of soiled litter during the task of cleaning out was less efficient and additional respiratory protection is therefore recommended for this procedure.
Detection, isolation and complete amino acid sequence of an aeroallergenic protein from rapeseed flour
Authors: MONSALVE R. I. , DE LA PEÑA M. A. GONZÁLEZ , LÓPEZOTÍÍN C. , FIANDOR A. , FERNÁNDEZ C. , VILLALBA M. , RODRÍGUEZ R.
Published: November 2003

Background
Seed proteins have been found to cause hypersensitivity by ingestion or inhalation. Rapeseed fiour was responsible for allergic symptoms in a patient, who develops into allergy to mustard spice.

Objective
To determine the presence of allergenic proteins in rapeseed fiour, and analyse the structure of the main component and its crossreactivity with the mustard allergeti.

Methods
SDS‐PAGE (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) and subsequent immunoblotting with a serum from a rapeseed allergic patient were performed to detect IgE‐binding proteins. Proteolytic digestiotis and high performance liquid chromatography were used to obtain the peptides from the allergenic BnIII napin from rapeseed flour. Automatic Edman degradations were carried out to determine their amino acid sequences, which were compared with other sequences in nucleotide and amino acid sequence databases. Crossreactivity assays were carried out by ELISA inhibition using sera from a rapeseed allergic patient and from patients allergic to mustard.

Results
The 2S albumins of rapeseed were recognized by the serum from a patient allergic to this seed. The most abundant isoform of the allergenic napins, Bnlll, was used for structural and immunological analysis. The protein consists of two different chains of 9.5 and 4,5 kDa. Their complete amino acid sequences were determined. The protein exhibited structural relationships with other napin‐like storage proteins from seeds. IgE and IgG crossreactivity between rapeseed and mustard allergens was also demonstrated. Considering the structural and immunological data, certain polypeptide regions are suggested to be involved in the allergenicity of these proteins.

Conclusions
Rapeseed contains 2S storage proteins which may cause allergy in hypersensitive individuals. These proteins exhibit great sequence similarity with 2S albumins from different seeds. Crossreactivity between mustard and rapeseed flours can be explained by sequetice homology.
Intranasal capsaicin is efficacious in non‐allergic, non‐infectious perennial rhinitis. A placebo‐controlled study
Authors: BLOM H. M. , VAN RIJSWUK J. B. , GARRELDS I. M. , MULDER P. G. H. , TIMMERMANS T. , VAN WIJK R. GERTH
Published: November 2003

Background
Several authors described capsaicin, the pungent substance in red pepper, as an efficacious therapy for non‐allergic non‐infectious perennial rhinitis (NANIPER). Repeated capsaicin application induces peptide depletion and specific degeneration of the unmyelinated sensory C‐fibres in the nasal mucosa.

Methods
We performed a placebo‐controlled (NaCl 0.9%) study with 25 NANIPER patients. Daily record charts and visual analogue scales (VAS) were used for clinical evaluation. Nasal lavages were obtained before, during, and after treatment.

Results
There was a significant and long‐term reduction in the VAS scores in the capsaicin group. No significant difference was found between the placebo and capsaicin treated groups for the mean group concentrations of leukotriene (LT) C4/D4/E4, prostaglandin D2 (PGD2). and tryptase. The levels of mast cell mediators, tryptase and PGD2. and leukotrienes, mediators derived from a variety of inflammatory cells, were low at baseline and comparable with levels observed in nasal lavages obtained from normals.

Conclusion
As involvement of inflammation could not be demonstrated, it is not surprising that capsaicin has no effect on inflammatory mediators. This suggests that inflammatory cells do not play a major part in the pathogenesis of NANIPER.
Selective type IV phosphodiesterase inhibitors prevent IL‐4‐induced IgE production by human peripheral blood mononuclear cells
Authors: COQUERET O. , BOICHOT E. , LAGENTE V.
Published: November 2003

Background
Selective type IV phosphodiesterase (PDE) inhibitors elicit anti‐inflammatory and bronchodilatory activities in vitro and in vivo which suggest that these drugs could provide a new therapeutic approach for asthma treatment.

Objective
Regarding the role of IgE production in allergic and inflammatory reactions of the airways, we investigated the effect of selective PDE inhibitors on IL‐4‐driven IgE production by peripheral blood mononuclear cells (PBMC) or by purified B lymphocytes.

Methods
PBMC or purified B lymphocytes from non‐allergic donors were stimulated for 13 days with IL‐4 (100U/mL) in the presence or in the absence of selective PDE inhibitors. IgE production is evaluated by an ELISA technique.

Results
The selective PDE IV inhibitors, rolipram and Ro 20–1724 (10μM), inhibit lL‐4‐induced IgE production by PBMC. but not by purified B lymphocytes. No modification of the IgE production was noted with the selective PDE III inhibitors, milrinone and SK&F94‐836, or the selective PDE V inhibitor, SK&F 96–231 (10 μM). Flow cytometry experiments showed that the effect of Rolipram could not be explained by the inhibition of the cell surface expression of the IL‐4 receptor. Similarly, no significant effect of PDE IV inhibitors was observed on PHA‐induced cell proliferation. The incubation of monocytes only with rolipram was sufficient to achieve a significant reduction of IgE production induced by IL‐4.

Conclusion
Taken together, these results indicate that PDE IV inhibitors reduce lL‐4‐induced IgE production by PBMC and suggest that the inhibition of IgE production could be explained by a failure of monocytes to provide the necessary costimulatory signals.
Thunderstorm‐related asthma – the epidemic of 24/25 June 1994
Authors: VENABLES K. M. , ALLITT U. , COLLIER C. G. , EMBERLIN J. , GREIG J. B. , HARDAKER P. J. , HIGHHAM J. H. , LATNG‐MORTON T. , MAYNARD R. L. , MURRAY V. , STRACHAN D. , TEE R. D.
Published: November 2003

Background
A large epidemic of asthma occurred following a thunderstorm in southern and central England on 24/25 June 1994. A collaborative study group was formed.

Objectives
To describe the epidemic and the meteorological, aerobiological and other environmental characteristics associated with it.

Methods
Collation of data from the Meteorological Office, the Pollen Research Unit, the Department of the Environtnent's Automatic Urban Network, from health surveillance by the Department of Health and the National Poisons Unit, from clinical experience in general practice and hospitals, and from an immunological study of some of the affected cases from north east London.

Results
The thunderstorm was a Mesoscale Convective System, an unusual and large form of storm with several centres and severe wind gusts. It occurred shortly after the peak grass pollen concentration in the London area. A sudden and extensive epidemic occurred within about an hour affecting possibly several thousand patients. Emergency services were stretched but the epidemic did not last long. Cases had high serum levels of IgE antibody to mixed grass pollen.

Conclusion
This study supports the view that patients with specific IgE to grass pollen are at risk of thunderstorm‐related asthma. The details of the causal pathway from storm to asthma attack are not clear. Case‐control and time series studies are being carried out.
Defective antigen processing associated with familial disseminated mycobacteriosis
Authors: D’Souza S , Levin M , Faith A , Yssel H , Bennett B , Lake R A , Brown I N , Lamb J R
Published: November 2003

To gain insights into a possible immune defect predisposing to disseminated mycobacteria infection, we studied three of six surviving children with disseminated Mycobacterium avium complex infection, who had no recognized form of immunodeficiency. We used mycobacteria isolated from the patients and diphtheria, tetanus and pertussis vaccine (DTP) to study antigen-specific T lymphocyte responses. We observed that interferon-gamma (IFN-γ) production by T cells in response to antigens (both mycobacteria and DTP) in these patients with disseminated infection was greatly impaired. This defect did not seem to be the result of T cell unresponsiveness, as phytohaemagglutinin (PHA) stimulation was able to induce high levels of IFN-γ comparable to those seen in control patients with localized infection. Further experiments showed that peripheral blood mononuclear cells (PBMC) from patients with disseminated infection were able to present influenza haemagglutinin (HA) peptides to specific T cell clones. However, this ability was lost when the whole HA protein was used as source of antigen. Taken together, these observations support the notion that the primary immune defect in these patients with disseminated mycobacterial infection rests in the antigen-processing functions of their antigen-presenting cells (APC). These findings may provide clues to the wider problem of susceptibility to mycobacteria and other intracellular pathogens and have implications in designing therapy for these patients.
Cytotoxic T lymphocytes specific for murine type II collagen do not trigger arthritis in B10 mice
Authors: Gao X-M , Mcmichael A J
Published: November 2003

To investigate the role of cytotoxic T lymphocytes (CTL) in arthritis, we set out to induce CTL specific for murine type II collagen (mCII) in a mouse model. The primary protein sequence of the murine pro-α1(II) was screened for fragments bearing H-2 Db or Kb binding motifs. Six fragments were identified and the corresponding peptides synthesized. One of these peptides, peptide P201 (amino acid 199–208 in the C-propeptide of the murine pro-α1(II)), was found to be a strong binder to H-2 Db. When used to treat RMA-S cells at 26°C, peptide P201 induced a four-fold increase of surface expression of H-2 Db. Administration of the P201-treated RMA-S cells into B10 mice (H-2b) induced strong CTL responses against the immunizing collagen peptide. Despite the high frequencies of mCII-specific CTL precursors in the periphery, however, the immunized mice showed no sign of arthritis up to 16 weeks after immunization. Implications of these data for autoimmunity and arthritis are discussed.
Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy
Authors: Wadhwa M , Bird C , Fagerberg J , Gaines-Das R , Ragnhammar P , Mellstedt H , Thorpe R
Published: November 2003

In this study, the development of neutralizing and non-neutralizing GM-CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli-derived GM-CSF and a colon carcinoma-reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM-CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM-CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM-CSF binding antibodies. However, only a proportion (40%) of the 19 antibody-positive patients developed antibodies that neutralized the biological activity of GM-CSF in an in vitro bioassay. The presence of GM-CSF neutralizing antibodies was associated with a significant reduction in GM-CSF-induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non-neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM-CSF preparations derived using different expression systems (chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross-react with endogenously produced GM-CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy.
Autoantibodies to the collagenous region of C1q occur in three strains of lupus-prone mice
Authors: Hogarth M B , Norsworthy P J , Allen P J , Trinder P K E , Loos M , Morley B J , Walport M J , Davies K A
Published: November 2003

We have developed an ELISA to measure murine autoantibodies to the collagenous region (CLR) of C1q, using the whole human C1q molecule as the solid-phase ligand, in the presence of 1 m NaCl. The assay was validated by testing positive sera from 20 mice using purified mouse C1q, and from 10 mice using purified human C1q-CLR, as the solid-phase ligands. There were highly significant correlations between results obtained with human C1q (whole molecule) and: (i) mouse C1q (rSp = 0.73, P < 0.001), and (ii) human C1q-CLR alone (rSp = 0.86, P = 0.001). Antibodies to C1q were measured in 53 MRL/lpr, 17 BXSB and 25 NZB/W lupus-prone mice. Median (range) anti-C1q (CLR) antibody levels in MRL/lpr, BXSB, and NZB/W autoimmune mice aged 3 months were 22 (16–66), 21 (17–39) and 19 (15–27) EU, respectively. The median anti-C1q antibody level in MRL/lpr mice aged 5 months was 76 (35–142) EU, significantly higher than that at 3 months (U = 558, P < 0.0005). Median anti-C1q antibody level in NZB/W mice at 8 months was 37 (13–74) EU and in BXSB mice at 11 months was 62 (31–231) EU, significantly higher than corresponding values at 3 months (U = 326, and U = 4, P < 0.001, respectively). This is the first demonstration of anti-C1q (CLR) antibodies in NZB/W and BXSB mice. The pathologic significance and the potential utility of these antibodies for monitoring disease in lupus-prone mice are under evaluation.
Memory lymphocytes from tuberculous effusions: purified protein derivative (PPD) stimulates accelerated activation marker expression and cell cycle progression
Authors: Lukey P T , Latouf S E , Ress S R
Published: November 2003

Accelerated PPD-specific proliferation and generation of CD4+ cytotoxic effectors by mononuclear leucocytes (MNL) from tuberculous effusions (EMNL) has been previously reported by our laboratory. In order to explore the contribution of the state of activation of MNL to accelerated reactivity, EMNL and peripheral blood (PB)MNL from seven patients with tuberculosis were assessed both ex vivo and after PPD stimulation. Flow cytometry revealed no difference in the activation state (IL-2 receptor and HLA-DR expression) or cell cycle progression ex vivo. However, CD4+CD29+ memory T cells were accumulated in EMNL compared with PBMNL. In vitro stimulation of EMNL with PPD resulted in accelerated expression of activation markers and progression through the cell cycle (peak after 4 days), whilst PBMNL exhibited normal activation kinetics (peak after 7 days). Accelerated reactivity could not be accounted for by quantitative differences in effusion CD4+CD29+ memory T cells compared with blood, but may be due to a qualitative difference in effusion memory T cells, which are shown to be in a post-activation state of differentiation. T cells entering S and G2/M phases of the cell cycle were largely of the activated memory phenotype. Activation marker expression occurred in association with up-regulation of CD4 antigen expression on the surface of EMNL. Thus accelerated expression of activation markers and cell cycle progression by CD4+CD29+ memory T cells may in part account for accelerated PPD reactivity in tuberculous effusions.
Antibodies to interferon (IFN) in hepatitis C patients relapsing while continuing recombinant IFN-α2 therapy
Authors: Antonelli G , Giannelli G , Currenti M , Simeoni E , del Vecchio S , Maggi F , Pistello M , Roffi L , Pastore G , Chemello L , Dianzani F
Published: November 2003

A number of trials have demonstrated that IFN-α is effective in chronic hepatitis C virus infection. It is known, however, that a number of chronic hepatitis C patients experience, after an initial response to IFN, disease reactivation or relapse (also called ‘breakthrough’) while IFN therapy is still ongoing. Since in a number of clinical conditions a significant correlation between development of antibodies to IFN and failure of therapy has been established, we addressed the possibility that the development of antibodies to IFN may take part in the relapse occurring in hepatitis C patients during recombinant IFN-α (rIFN-α) therapy. The prevalence of neutralizing (NA) and binding antibodies (BA) to rIFN-α2 has been evaluated in 45 patients with chronic hepatitis C treated with rIFN-α2a who first normalized aminotransferase (ALT) levels, and subsequently showed disease reactivation while on treatment. The presence of NA and BA was tested before therapy, during the response to IFN treatment, and at the time when ALT started to rise again to abnormal levels. The results showed that no patients had detectable antibodies to IFN before therapy and during the period of response to the therapy, while most of them (88.9%) developed NA and/or BA to IFN-α2 concomitantly with disease reactivation. In particular, in 29 of the 45 patients (64.4%) ALT normalized on treatment and rose to abnormal levels when NA appeared in their serum, while in 11 of the 16 (68.8%) remaining patients the relapse was associated with BA development. The frequency of seroconversion in these patients is significantly higher than that observed in the control group. These data indicate that antibodies to IFN may be responsible for breakthrough in the majority of patients showing disease reactivation while rIFN-α therapy is still ongoing.
Inhibitory effect of clarithromycin on costimulatory molecule expression and cytokine production by synovial fibroblast-like cells
Authors: Matsuoka N , Eguchi K , Kawakami A , Tsuboi M , Kawabe Y , Aoyagi T , Nagataki S
Published: November 2003

This study was undertaken to investigate the immunomodulatory effect of clarithromycin against synovial fibroblast-like cells (synoviocytes). Synovial tissue obtained from rheumatoid arthritis (RA) or osteoarthritis (OA) patients was enzymatically digested to separate synoviocytes. The synoviocytes were cultured with or without cytokines in the presence of various concentrations of clarithromycin. The expression of costimulatory molecules was examined on the surface of the synoviocytes, using specific MoAbs and flow cytometry. The production of cytokines by synoviocytes was also measured using an immunoenzymatic assay. Finally, autologous T cells were stimulated by interferon-gamma (IFN-γ)-treated synoviocytes in response to purified protein derivative (PPD). In some experiments, MoAbs specific for costimulatory molecules or clarithromycin were added and 3H-thymidine incorporation was counted. Intercellular adhesion molecule-1 (ICAM-1), LFA-3 and vascular cell adhesion molecule-1 (VCAM-1) were detected on the surface of both RA and OA synoviocytes. However, ICAM-2, B7–1 and B7–2 were not detected, and cytokines failed to induce these molecules. Both spontaneous and up-regulated expression of ICAM-1, LFA-3 and VCAM-1 by IFN-γ, IL-1β or 12-o-tetradecanoyl phorbol 13-acetate (TPA) were markedly suppressed by clarithromycin in a dose-dependent manner at concentrations between 0.1 and 10 μg/ml. The production of IL-1β, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-1α and tumour necrosis factor-alpha (TNF-α) by synoviocytes was detected. Clarithromycin significantly suppressed the production of these cytokines, but did not enhance IL-10 production. Finally, autologous T cells were stimulated by IFN-γ-treated synoviocytes in response to PPD. As clarithromycin suppressed HLA-DR and costimulatory molecule expression was enhanced by IFN-γ, autologous T cell proliferation was markedly inhibited by clarithromycin. Clarithromycin has a considerable immunosuppressive effect on synoviocytes by inhibiting costimulatory molecule expression, cytokine production and antigen-specific T cell proliferation induced by synoviocytes.
Definition of three minimal T helper cell epitopes of rubella virus E1 glycoprotein
Authors: Marttila J , Ilonen J , Lehtinen M , Parkkonen P , Salmi A
Published: November 2003

To characterize T cell-recognized epitopes on rubella virus (RV) E1 glycoprotein, IL-2-dependent RV-specific T cell lines were established from 14 rubella-seropositive healthy donors. The responses of these lines were studied by using a panel of 94 partially overlapping synthetic peptides of 15 amino acids (aa) length covering the known nucleotide sequence of RVE1 glycoprotein. Two to seven peptide-defined epitopes were recognized by the T cell lines, but a large interindividual variation was found. T cell reactivity was most often localized to the regions between aa 276 and 290, aa 381 and 395 and aa 410 and 420. Analysis of overlapping, truncated peptides revealed three minimal T helper cell epitopes VIGSQARK, KFVTAALLN and RVIDPAAQ in aa positions 280–287, 385–393 and 412–419, respectively.
Human monoclonal rheumatoid factors augment arthritis in mice by the activation of T cells
Authors: Ezaki I , Okada M , Yoshikawa Y , Fujikawa Y , Hashimoto M , Otsuka M , Nomura T , Yamamoto K , Watanabe T , Shingu M , Nobunaga M
Published: November 2003

In order to investigate the in vivo role of rheumatoid factor (RF), the effects of the administration of human monoclonal (m) IgM-RF and IgG-RF on the development of arthritis in mice were examined. The administration of human mRFs into mice immunized with type II collagen (CII) markedly enhanced the clinical score and paw swelling. The severity of arthritic joint disease with a marked infiltration of lymphoid cells, proliferation of synovial membrane, pannus formation and destruction of articular cartilage was significantly enhanced in both groups receiving RF (RF-enhanced arthritis). Skin ulcers were also observed in some of these RF-enhanced arthritis mice, whereas no such signs were observed in CII-immunized mice without mRFs. Both IgM-RF and IgG-RF increased CII-specific IgG antibodies in circulation, and the severity of arthritis correlated with the production of high titres of anti-CII antibodies. In vivo treatment of RF-enhanced arthritis mice with an anti-CD4 MoAb or an anti-CD8 MoAb inhibited the induction and progression of arthritis in these mice. Administration of RF to severe combined immunodeficient (SCID) mice with arthritis developed by the transfer of spleen cells from CII-immunized mice, prolonged the arthritis and enhanced the severity. This murine model of RF-enhanced arthritis may provide a useful tool for analysing the pathogenesis of rheumatoid arthritis in RF-positive patients.
Antibody cross-reactivity induced by the homologous regions in glutamic acid decarboxylase (GAD65) and 2C protein of coxsackievirus B4
Authors: Lönnrot M , Hyöty H , Knip M , Roivainen M , Kulmala P , Leinikki P , Åkerblom H K ,   Childhood Diabetes in Finland Study Group
Published: November 2003

GAD65 contains an amino acid sequence which is highly homologous with a sequence in the 2C protein of coxsackievirus B4 (CBV4-2C). In the present study the possibility that this region could contain an epitope capable of inducing immunological cross-reactivity between CBV4-2C and GAD65 was evaluated. Six out of seven rabbit sera, which were raised against seven different synthetic peptides carrying various modifications of the homology sequence, showed cross-reactivity between 2C, GAD65 and GAD67 derived peptides in ELISA. There was substantial cross-reactivity between 2C and GAD65 peptides, but not between 2C and GAD67 peptides. The most cross-reactive peptides were those corresponding to the 2C sequences FIEWLKVKILPEVKEK and KILPEVKEKHEFLSRL. When the binding of the four 2C peptide-specific sera to the GAD65 protein was analysed in immunoprecipitation, two sera were found to be cross-reactive (anti-FIEWLKVKILPEVKEK and anti-WLKVKILPEVKEKHEF). One of these (anti-WLKVKILPEVKEKHEF) reacted also with coxsackie B virus (CBV)-infected cells. Antibodies against this epitope were induced during enterovirus (including CBV) infections in initially healthy children who later progressed to clinical insulin-dependent diabetes mellitus (IDDM). Antibody responses were frequent also in constantly GAD65 antibody-negative non-diabetic children, and antibody levels did not differ between newly diagnosed IDDM patients and matched control subjects. Blocking experiments confirmed that the observed reactivity of both rabbit and human antibodies was immunologically specific. The results suggest that the epitope is antigenically highly similar in 2C and GAD65, and that peptide immunization induces antibodies which cross-react with these molecules. However, the significance of this phenomenon in the pathogenesis of IDDM remains to be confirmed, as the peptide antibody levels were similar in patients with recent-onset IDDM and in control subjects.
Role of IL-4 in delayed type hypersensitivity
Authors: Asherson G L , Dieli F , Sireci G , Salerno A
Published: November 2003

IL-4 plays a key role in the contact sensitivity skin reaction. This has several implications. First, the view that contact sensitivity (CS) is only mediated by cells with a Th1 profile of cytokine secretion needs modification, in the light of the essential role of IL-4 at the effector stage. Second, the concept of a single cell involved in the systemic transfer of CS is no longer tenable, as it is known that both αβ and γδ cells are required. Studies with the cell lines (which contain both αβ and a few γδ cells) suggest that this double requirement may involve the action of IL-4 on γδ cells, which bear receptors for IL-4. Finally, the view that T cell lines only transfer CS when injected locally, but not when injected intravenously (systemic transfer), is correct but incomplete, as T cell lines actually give systemic transfer of CS, providing the cell line or the recipient is treated with IL-4.
Increased mucosal synthesis of rheumatoid factor (RF) in coeliac disease
Authors: Hällgren J , Knutson F , Lavö B , Hällgren R
Published: November 2003

Increased serum levels of RF have been reported in patients with gluten sensitivity. The objective of this study was to investigate the in vivo secretion of different isotypes of RF in the small bowel in coeliac disease. Nineteen patients were investigated by perfusion of a defined jejunal segment, and the jejunal perfusion fluid was analysed for the presence of IgA and IgM anti-Fc (IgG). Five of the patients studied had serum IgA deficiency. Patients with partial/subtotal villous atrophy but no IgA deficiency (n = 7) had a four-fold increase of IgM-RF (P<0.001) and a three-fold increase of IgA-RF (P<0.001) compared with healthy controls (n = 29). Patients with normal jejunal mucosa but no IgA deficiency (n = 7) had similar IgA-RF and IgM-RF concentrations to healthy controls. Patients with serum IgA deficiency had no IgA-RF detectable in jejunal fluid but the highest IgM-RF concentrations, in particular in active disease. The coeliac patients had serum levels of IgA-RF and IgM-RF within the reference ranges. Jejunal fluid levels of IgA-RF and IgA-anti-gliadin antibodies were significantly correlated (P<0.001). The data indicate that enhanced jejunal mucosal production of RF occurs above all in active coeliac disease. The findings suggest that the immune response to gluten induces a mucosal RF synthesis.
Inverted Vδ1/Vδ2 ratio within the T cell receptor (TCR)-γδ T cell population in peripheral blood of heart transplant recipients
Authors: Vaessen L M B , Schipper F , Knoop C , Claas F H J , Weimar W
Published: November 2003

We investigated the levels of TCR-γδ T cells and their subpopulations Vδ1 and Vδ2 in the peripheral blood lymphocytes (PBL) of 28 heart transplant (HTx) patients. Patients (n = 10) receiving cyclosporin A (CsA) for treatment of a nephrotic syndrome (NS) and 10 healthy individuals served as controls. There was no difference in levels of TCR-γδ T cells between the different groups. However, an elevated proportion of Vδ1+γδ T cells was found in the PBL of HTx patients, especially when these cells were present in their graft-infiltrating lymphocyte (GIL) cultures. Vδ1+γδ T cells of HTx patients showed normal expression of CD45RO and lacked the activation markers CD25 and HLA-DR. After expanding in IL-2-containing medium, PBL cultures of HTx patients more often were dominated by Vδ1 cells than PBL cultures of controls, in which Vδ2 cells were predominantly grown. The aberrant composition of the TCR-γδ population in HTx patients was not a result of immunosuppressive medication, since the proportion Vδ1+γδ T cells was normal in the PBL of the NS patients receiving a similar dose of CsA. It is postulated that long-term antigenic stimulation by the graft, at low level, might be responsible for the altered composition of the γδ pool in the HTx patients. Since no donor HLA-specific γδ T cells have been detected, other ligands, such as heat shock proteins, may be involved.
Prevalence of hepatitis C in patients with primary antibody deficiency
Authors: Webster A D B , Brown D , Franz A , Dusheiko G
Published: November 2003

Concern over sporadic hepatitis C virus (HCV) infection from treatment with contaminated immunoglobulin in patients with immunodeficiency prompted us to screen serum from 44 patients for HCV RNA. We found no evidence of either horizontal or iatrogenic infection in our Clinic, except in two patients known to have received contaminated batches, and in one who had had regular plasma infusions. Thus, common variable immunodeficiency (CVID) patients in England do not have a high incidence of HCV infection despite exposure to various immunoglobulin products. This contrasts with the recently reported high prevalence in similar patients in Italy. Although our data reinforce the impression that both intramuscular and current licensed i.v. preparations in the UK are safe, manufacturers should be encouraged to PCR screen all batches of immunoglobulin for HCV.
Normal levels of soluble E-selectin, soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1) decrease with age
Authors: Nash M C , Wade A M , Shah V , Dillon M J
Published: November 2003

sE-selectin, sICAM-1, sVCAM-1 and von Willebrand factor (vWF) were assayed in 238 samples in a longitudinal study of 81 normal children from 9.5 to 15.5 years old. Multilevel modelling was used to quantify changes with age. sE-selectin, sICAM-1 and sVCAM-1 all fell significantly over the age range (by 17%, 16%, and 10%, respectively). In contrast, levels of vWF were not age-dependent. Our findings highlight the need for age-matched controls when studying cell surface adhesion molecules in disease groups, and may imply developmental changes in expression of these molecules and their shedding from the cell surface.
Hepatitis B virus (HBV)-specific in vitro antibody production by peripheral blood mononuclear cells (PBMC) after vaccination by recombinant hepatitis B surface antigen (rHBsAg)
Authors: Ducos J , Bianchi-Mondain A-M , Pageaux G , Conge A M , Poncet R , Vendrell J P , Segondy M , Serre A
Published: November 2003

To study the immunization induced by rHBsAg, we analysed the in vitro antibody production (IVAP) to HBsAg by PBMC from 18 subjects vaccinated by two injections on days 0 and 30. HBsAg-specific IVAP was detectable in all subjects after both the first and the second injection, and lasted for about 10 days and then disappeared. However, when the spontaneous HBsAg-specific IVAP became negative, HBsAg stimulation of PBMC cultures induced again a specific HBsAg IVAP. Cultures of cell populations separated by erythrocyte rosetting or Percoll density centrifugation showed that the cells responsible for spontaneous secretion, after in vivo stimulation, were low-density B lymphocytes. High-density B lymphocytes were involved in anti-HBs production induced by in vitro stimulation when spontaneous secretion disappeared. These data suggest that the IVAP test could be a source of important information along with serologic analysis for exploration of the immune response to hepatitis B vaccine.
Experimental T cell-mediated inflammatory reactions in the murine oral mucosa. II. Immunohistochemical characterization of resident and infiltrating cells
Authors: Ahlfors E , Jonsson R , Czerkinsky C
Published: November 2003

The nature and phenotype of infiltrating cells in DTH-like reactions elicited in the murine oral mucosa have been examined by routine histological and immunohistochemical procedures. During the first few hours that followed buccal challenge with the contact sensitizer oxazolone, a discrete lymphocytic reaction was disclosed in the oral mucosa of animals previously sensitized at skin sites, but was absent in animals that had been sensitized at buccal sites. The early lymphocytic reaction in the oral mucosa of skin-sensitized animals preceded the emergence of CD11+ polymorphonuclear cells (PMN) which was most prominent 8 h after hapten challenge and invaded the whole thickness of the oral epithelium. The PMN rapidly disappeared by 24 h. In contrast, early PMN infiltration was virtually absent in specimens from animals similarly challenged but that had been sensitized at local buccal sites. Irrespective of site of initial sensitization, inflammatory reactions developed in the oral mucosa, being maximal by 24 h. At that stage, CD11+ macrophages were the predominant cell type. Both CD4 and CD8 T lymphocytes were scattered in both lamina propria and epithelium, and their numbers were raised throughout the time period studied (2–168 h). IL-2 receptor expression was maximal 16 h post-challenge, and was paralleled by increased DNA synthesis in CD4+ and CD8+ cells, as demonstrated by paired immunohistoautoradiography. Focal accumulations of mononuclear cells containing IL-2-producing cells were readily detected as early as 2–3 h following local challenge with hapten in animals primed at skin but not at buccal sites. Maximal IL-2 staining was detected at 24 h irrespective of initial sensitization site. Interferon-gamma-producing cells were detected at 8 h post-challenge and remained increased during the first 24 h. MHC class II expression was detected on few oral mucosa cells during the first 4 h following hapten challenge, being mainly confined to dendritic-like cells. Consistent with increased numbers of macrophages, MHC class II expression was most intense in specimens obtained 8–24 h after hapten challenge. Thereafter, MHC class II expression was still observed in specimens obtained as late as 72 h, but was essentially associated with patches of basal keratinocytes. Taken together, these observations support the notion that the murine oral mucosa can serve as the site of expression of locally or remotely induced DTH reactions, but also indicate that the site of initial sensitization can profoundly affect the cellular composition of inflammatory reactions subsequent to local buccal challenge.
Successful induction of severe destructive arthritis by the transfer of in vitro-activated synovial fluid T cells from patients with rheumatoid arthritis (RA) in severe combined immunodeficient (SCID) mice
Authors: Sakata A , Sakata K , Ping H , Ohmura T , Tsukano M , Kakimoto K
Published: November 2003

In order to investigate the role of pathogenic T cells in RA, the establishment of an RA model using patients’ T cells is thought to be essential. In this study, multiple and severe destructive arthritis was established by transferring in vitro-stimulated synovial fluid T (SFT) cells from patients with RA through simultaneous injection into knee joint and peritoneal cavity of SCID mice without causing xenogeneic graft-versus-host disease (GVHD). Neither the transfer of unstimulated SFT cells nor sole i.p. injection was sufficient to induce severe arthritis. Interestingly, in contrast with SFT cells, in vitro-activated peripheral blood lymphocytes from RA patients failed to trigger such arthritis, suggesting that pathogenic T cells might be concentrated in synovial fluid of RA patients. This, the first severe arthritis model mimicking RA induced by RA patients’ T cells, is expected to provide important information about RA pathogenesis and a possible therapeutic approach.
Ivermectin-induced immunopotentiation in onchocerciasis: recognition of selected antigens following a single dose of ivermectin
Authors: Akuffo H , Maasho K , Lavebratt C , Engström K , Britton S
Published: November 2003

Onchocerciasis is associated with blindness and gross skin changes, believed to be a consequence of the immune response to antigens released from the offspring of the female worm of Onchocerca volvulus, the microfilariae (mf). An effective microfilaricidal drug is now available which quickly reduces the mf burden without affecting the adult worm. There exist foci in onchocerciasis endemic areas where some of the patients have many mf in their skin but relatively few clinical symptoms. This state of hyposensitivity is believed to be due to immunosuppression. The aim of this study was to address the question of the basis of, and the effect of ivermectin treatment on this immunosuppression. Female adult worms of O. volvulus were used as whole or fractionated antigens to stimulate peripheral blood mononuclear cells. Microfilariae are found in the reproduction tract of the female worms, and thus an antigen preparation of the female adult O. volvulus contains both exclusive adult antigens as well as antigens from microfilariae. Cells were obtained from onchocerciasis patients, individuals of similar socio-economic status living in the same Ghanaian village, but who showed no parasitological or clinical evidence of onchocerciasis (exposed endemic controls), healthy Ghanaians living in areas where transmission of onchocerciasis does not seem to occur (non-exposed endemic controls) and unexposed healthy Swedish donors. As a group, cells from onchocerciasis patients proliferated to a lesser degree than cells from the exposed endemic control and the non-exposed endemic control groups to the whole worm antigen, whereas the phytohaemagglutinin (PHA) response was strongest in the patients. Proliferative responses of above 1000 ct/min to fractions of the worm extract were only evident in the cells from a few individuals in each of the various groups. However, 28 days following ivermectin treatment, cells from all onchocerciasis patients were able to mount significantly enhanced proliferation to a fraction of approximately 96 kD (fraction 3), while only four of nine of this group showed an increased response to the whole worm antigen. The proportional increase in the response to the whole organism in these individuals was of a much lower magnitude than the increased response to fraction 3. The O. volvulus antigen-specific immunosuppression observed in these onchocerciasis patients appears to be due to suppressive antigens which have the capacity to mask the potential response to selected antigens of O. volvulus, and ivermectin treatment possibly modulates the immune response, allowing for stepwise recognition of such antigens. Since ivermectin treatment kills only the microfilariae and not the adult worm, the putative suppressive antigens would be expected to be from the microfilariae.
Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis
Authors: Roche P W , Winter N , Triccas J A , Feng C G , Britton W J
Published: November 2003

Proteins secreted across the cell wall of mycobacteria are important antigens recognized early in the host response to mycobacterial infection. MPT64 is a 23-kD secreted protein restricted to members of the Mycobacterium tuberculosis complex which elicits T cell responses and cutaneous DTH reactions in Myco. tuberculosis-infected animals. Patients with tuberculosis and their tuberculin-positive contacts respond to the protein, but recipients of bacille Calmette–Guérin (BCG) vaccine strains lacking the mpt64 gene do not. In the present study, we describe the development of a unique recombinant mycobacterial vector which secretes the encoded Myco. tuberculosis protein MPT64 at high levels into the culture filtrate, from which the protein is isolated by a single-step affinity chromatographic step. The purified protein was recognized by both polyclonal and monoclonal anti-MPT64 antibodies. The T cell reactivity of the protein was confirmed by its ability to stimulate human anti-rMPB64 T cell lines. The Myco. smegmatis recombinant MPT64 protein was superior to the Escherichia coli rMPB64 protein, which has identical amino acid sequence, in eliciting cutaneous DTH reactions in guinea pigs sensitized with Myco. tuberculosis. Animals sensitized with BCG strains lacking the mpb64 gene failed to respond to MPT64. Similarly, interferon-gamma (IFN-γ) responses in tuberculosis patients and their contacts were higher to the Myco. smegmatis form of the protein. The potential of this form of the Myco. tuberculosis MPT64 protein as a skin test reagent for tuberculosis is discussed.
Differential expression of Fc receptors for IgG by monocytes and granulocytes from neonates and adults
Authors: Maeda M , van Schie R C A A , Yüksel B , Greenough A , Fanger M W , Guyre P M , Lydyard P M
Published: November 2003

The immature neonatal immune system is thought to result in increased risk of infection. Receptors for the Fc moiety of IgG (FcγR) are important in antibody-mediated clearance of microbes by granulocytes and monocytes/macrophages. As an approach to understanding their role in neonatal life, we have compared the constitutive expression of the three Fc receptors—FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)—by neonatal and adult blood monocytes and granulocytes using quantitative immunofluorescence by flow cytometry. Our results confirm that there is a small subpopulation of FcγRII-positive monocytes in adult blood, and furthermore show that this is absent or at a low percentage in cord blood samples. However, the main population of cord blood monocytes expresses low, but significantly higher levels of FcγRIII than adult monocytes. No differences were seen in the quantitative expression of FcγRI and FcγRII. Neonatal granulocytes expressed significantly higher levels of both FcγRI and FcγRII but significantly lower levels of FcγRIII. The data are discussed in terms of the possible role of cytokines and susceptibility to infection.