bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of antibodies to ovarian antigens in women with premature ovarian failure
Authors: WHEATCROFT N J , TOOGOOD A A , LI T C , COOKE I D , WEETMAN A P
Published: July 2010

Premature ovarian failure is a common condition of uncertain aetiology in most cases, although autoimmunity is thought to play a role in a proportion of eases. The frequency of ovarian antibodies, which may be markers for an autoimmune aetiology in this condition, remains unclear. To define this further, we have examined the sera of 45 women with premature ovarian failure (five with iatrogenic ovarian failure, nine with an associated autoimmune disease, and 27 with idiopathic ovarian failure), as well as four women with infertility due to Turner's syndrome and 41 pre- and post-menopausal controls. Using two human ovarian antigen preparations, 24% and 60% of the ovarian failure patients reacted in an ELISA (P < 0·05 and P < 0·001 compared with controls), but frequent cross-reactivity was found with fallopian tube antigens. The apparent aetiology of ovarian failure did not correlate with the presence of ovarian antibodies. Using bovine ovary as an antigen, there was a significant overall increase in binding by the ovarian failure patients, but this was almost identical to binding in an ELISA with bovine fallopian tube. In contrast to a previous report, there was no significant increase of binding to soluble or Triton-extracted membrane fractions of bovine corpora lutea containing the LH/hCG receptor by the patients with ovarian failure. These results suggest that ovarian antibodies are common in premature ovarian failure, but their specificity and pathogenic role are questionable.
Characterization of EN4 monoclonal antibody: a reagent with CD31 specificity
Authors: BURGIO V L , ZUPO S , RONCELLA S , ZOCCHI M , RUCO L P , BARONI C D
Published: July 2010

EN4 MoAb was originally described as a MoAb that reacts specifically with human endothelial cells, and the reagent was not assigned to any of the presently known CD. Here, we provide evidence indicating that EN4 reacts with the CD31 antigen. Thus, EN4 stains strongly murine fibrobtasts transfected with the human CD31 gene. Furthermore, SDS-PAGE analysis of immunoprecipitates of cell lysates from surface-iodinated Jurkart T cells demonstrated that EN4 and reference CD31 MoAb recognized the same antigen, of 130 kD mol. wt. Finally, both EN4 and CD31 gave the same pattern of reactivity when tested on tonsillar or peripheral blood lymphoid cells by FACS analysis or by immunohistochemistry on sections of a variety of human tissues. EN4, however, proved consistently more efficient than the reference anli-CD3l MoAb as judged by both the intensity of fluorescence or of tissue staining. This property has thus allowed a better characterization of the tissue and cellular distribution of CD31.
Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin
Authors: PALL A A , VARAGUNAM M , ADU D , SMITH N , RICHARDS N T , TAYLOR C M , MICHAEL J
Published: July 2010

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0·016 10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab′)2: fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0·002 2·65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab′)2 of normal human IgG from individual donors (1·8–12·2% al the maximum concentration of 2 mg/ml). F(ab′)2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab′)2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab′)2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab′)2 fragments of PHIG bind to F(ab′)2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.
The significance of blood levels of IgM, IgA, IgG and IgG subclasses in Sudanese visceral leishmaniasis patients
Authors: ELASSAD A M S , YOUNIS S A , SIDDIG M , GRAYSON J , PETERSEN E , GHALIB H W
Published: July 2010

We developed an ELISA test using leishmania antigenic extracts to detect antigen-specific antibody responses, including subclass and isotype analysis, in visceral leishmaniasis (VL) patients from the Sudan. A total of 92 parasitologically proven patients were compared with cutaneous leishmaniasis, schistosomiasis, malaria, onchocerciasis and tuberculosis patients, as well as with healthy endemic and non-endemic controls. Some VL patients were examined before and after chemotherapy. VL patients showed significantly higher IgG responses compared with all other groups (93·4% sensitivity, 93·7% specificity), and higher (but not significantly) IgM responses. All groups showed low IgA levels. All groups showed low IgA levels. All IgG subclasses, IgG1, 2, 3, and 4, showed higher levels in patients than all other groups, with IgG1 and IgG3 levels being significantly reduced following treatment. The rank order for specificity and sensitivity for IgG subclasses was IgG3 > IgG I> IgG2> IgG4.
High levels of circulating IL-10 in human malaria
Authors: PEYRON F , BURDIN N , RINGWALD P , VUILLEZ J P , ROUSSET F , BANCHEREAU J
Published: July 2010

IL-10 is a monocyte/lymphocyte derived cytokine which has been shown to inhibit certain cellular immune responses such as delayed hypersensitivity. In particular, the production of tumour necrosis factor (TNF), IL-I and IL-6, which are involved in malaria pathology, are strongly inhibited by IL-10. Accordingly, we examined whether IL-10 could be involved in a human acute parasitic infection such as Plasmodium falciparum malaria. Human IL-10 levels in plasma were determined by two-site ELISA method, taking care to avoid non-specific reactions due to autoantibodies. Fourteen cerebral, 11 severe, and 20 mild malaria cases had mean IL-10 levels of 2812, 2882 and 913 pg/ml, respectively, while 98% of healthy individuals had undetectable (less than 100 pg/ml) circulating IL-10. Thirteen of the 25 cerebral/severe cases had >2000 pg/ml. In 11 hospitalized patients, circulating IL-10 levels were found to return to virtually normal levels 7 days after antimalarial chemotherapy when biological and clinical malaria features had disappeared (mean levels fell from 3880 to 333 pg/ml). Further studies are required to determine whether these elevated levels of IL-10 play a beneficial role by reducing the parasite-induced inflammatory response, or a detrimental one by decreasing the cellular immune responses.
C6 haplotypes: associations of a Dde I site polymorphism to complement deficiency genes and the Msp I restriction fragment length polymorphism (RFLP)
Authors: FFRNIE B A , HOBART M J , DELBRIDGE G , POTTER P C , ORREN A , LACHMANN P J
Published: July 2010

Complement C6 has a common charge polymorphism designated A and B with gene frequencies of 0·65 and 0·35. The probable molecular basis for this is a Glu (C6A) for Ala (C6B) substitution at amino acid position 98, and is detected by digestion with the restriction enzyme Dde I of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA. C6A was found to be Dde I-positive and C6B corresponds to Dde I-negative. We have applied out Dde I A/B polymorphism genotyping method to the investigation of C6-delicient individuals with complete (C6Q0) and sub-total deficiency (C6SD) protein phenotypes, including members of four families. We have also investigated the RFLP detected by digestion of genomic DNA with the enzyme Msp I, which is due to a polymorphic site located in the 5′ section of the gene, the variable sequence of which has yet to be determined. Sixteen out of seventeen unrelated C6Q0 subjects were found to be genotypically Dde I B/Msp I-negative: the remaining subject was heterozygous at both the loci under investigation. The C6SD phenotype was found to be associated with the Dde I A/Msp I-positive genotype in two families with combined C6/C7 subtotal deficiency and two with C6SD. It can be concluded that the two forms of C6 deficiency, C6Q0 and C6SD, arose independently on two different C6 allelic backgrounds. These associations have allowed the genotyping of the rare families that contain both types of deficiency. We have also defined a number of normal C6 Dde I/Msp I haptotypes in Caucasians and Cape Coloured populations.
Mature helper T cell requirement for immunoglobulin production by neonatal naive B cells injected intraperitoneally into severe combined immunodeficient (SCID) mice
Authors: HASUI M , MIYAWAKI T , ICHIHARA T , NIIDA Y , IWAI K , YACHIE A , SEKI H , TANIGUCHI N
Published: July 2010

It is accepted that human neonatal naive B cells produce mainly IgM in vivo as well as in vitro. Our previous work has demonstrated that i.p. injection of neonatal B cells together with adult mature T cells induces substantial levels of human IgG in the serum of SCID recipient mice. The present study was further attempted to determine the cellular components required for immunoglobulin production by neonatal B cells in SCID mice. When neonatal B and adult T cells were transferred into the SCID mice, human immunoglobulins, largely of IgG, were maximally detected in the serum around 6 weeks after a cell transfer. Depletion of CD4+ T cells from adult T cells resulted in undetectable levels of human immunoglobulin in the serum. By contrast, CD4+ T cell-enriched populations exhibited an enhancing effect on immunoglobulin production by neonatal B cells. Higher levels of immunoglobulin, including IgA and IgM, were detected in the peritoneal fluid than in the serum as early as 2 weeks after the cell transfer. Human T cells expressing activation antigens such as CD45RO and HLA-DR antigens were identified in the peritoneal lavages. These results suggest that neonatal naive B cells are able to differentiate into cells producing all classes of immunoglobulin in the presence of mature CD4+ T cells in a SCID mouse environment. The peritoneal cavity of SCID mice appears to provide a suitable place for immune responses by human cells, possibly in association with a certain xenogeneic reaction.
Expression of human T lymphotropic virus type 1 (HTLV-1) tax/rex gene in fresh bronchoalveolar lavage cells of HTLV-1-infected individuals
Authors: HIGASHIYAMA Y , KATAMINE S , KOHNO S , MUKAE H , HINO S , MIYAMOTO T , HARA K
Published: July 2010

Accumulating evidence has suggested the involvement of HTLV-1 in the inflammatory lesions of various organs, including the lung. However, the causal relationship between HTLV-1 and inflammatory responses in the organs remains to be elucidated. In order to evaluate the expression of HTLV-1 and its effects in the lung, we examined the expression of mRNA for the HTLV-1 tax/rex gene in fresh bronchoalveolar lavage cells (BALC) and peripheral blood mononuclear cells (PBMC) of 23 seropositive individuals, including six patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), by use of an improved method of reverse transcription-polymerase chain reaction (RT-PCR). The tax/rex mRNA was more frequently detected in BALC than in PBMC. All the HAM/TSP patients and eight of 17 carriers without neurological symptoms showed the expression of tax/rex mRNA in the BALC. IgM class antibodies to HTLV-1 were preferentially detected in sera of the tax/rex mRNA-positive individuals. The detection of tax/rex mRNA correlated closely with the presence of lymphocytosis accompanied by an elevated proportion of IL-2 receptor-bearing T cells in the BALC. Our findings indicate the crucial role of viral expression in the inflammatory response in the lung in HTLV-1-infected individuals.
Active immunization by a dengue virus-induced cytokine
Authors: CHATURVEDI U C , MUKERJEE R , DHAWAN R
Published: July 2010

Dengue type 2 virus (DV)-induced cytotoxic factor (CF) is capable of reproducing various pathological lesions in mice that are seen in human dengue. The present study was undertaken to investigate the protective effect of active immunization of mice with CF, Mice were immunized with 5 μg of CF and prevention of CF-induced increase in capillary permeability and damage to the blood brain barrier were studied at weekly intervals, up to 48 weeks, by challenging with 3 μg of CF, Maximum protection against increase in capillary permeability and damage to the blood-brain barrier was observed in week 4 after immunization. A breakthrough in the protection occurred with higher doses of CF in a dose-dependent manner. Challenge with a lethal intracerebral (i.e.) dose of DV showed significantly prolonged mean survival time and delayed onset of symptoms of sickness in the immunized mice compared with the normal mice, but the titre of the virus in the brain was similar in the two groups. On i.p. challenge with the virus the protection against damage to the blood-brain barrier was 86±7% at week 4 and 17±4% at week 26 after immunization. Sera obtained from the immunized mice showed the presence of CF-specific antibodies by ELISA, Western blot, and by neutralization of the cytotoxic activity of CF in vitro. The present study describes successful prevention of a cytokine-induced pathology by specific active immunization.
Circulating adhesion molecules in sarcoidosis
Authors: HAMBLIN A S , SHAKOOR Z , KAPAHI P , HASKARD D
Published: July 2010

Sarcoidosis is a disease of unknown etiology characterized by non-caseating granulomata together with a number of systemic abnormalities. We have recently shown these include increased expression of the integrins CD11/CD18 on peripheral blood leucocytes. Here we have measured serum levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1) in 23 patients and 14 normal controls using antigen capture sandwich ELISAs. Median circulating E-selectin levels in the patients were nearly three times those of the controls (P<0·0001, Mann-Whitney U-test), whilst ICAM-I but not VCAM-1 levels were only slightly elevated. These results show that endothelial cell activation and shedding of E-selectin into the circulation are additional features of the pathology of sarcoidosis.
Establishment of a monoclonal antibody to plasma cells: a comparison with CD38 and PCA-1
Authors: HATA H , MATSUZAKI H , MATSUNO F , SONOKI T , TAKEMOTO S , KURTBAYASHI N , NAGASAKI A , TAKATSUKI K
Published: July 2010

A new monoclonal antibody which recognizes plasma cells was developed by utilizing two myeloma cell lines, KMS12PE (12PE) and KMS12BM (12BM). established from the pleural of fusion and bone marrow, respectively, of the same patient. Since I2BM expresses CD20, CD38, and PCA-I antigens, while 12PE has lost CD20. 12PE is considered to be phenotypically more mature than 12BM. The 12PE cells were used to immunize a BALB/c mouse and a MoAb was produced which was more reactive to 12PE than to 12BM. Thus, a clone, D2, was obtained. On Western blotting, D2 detected a single band of 54 kD under both reduced and non-reduced conditions. This antigen was not detected by Western blotting in peripheral blood lymphocytes that had been stimulated with pokeweed mitogen (PWM) for 7 days or in those not so stimulated. On flow cytometry, D2 detected a myeloma cell line, RPMI 8226. Another myeloma cell line, U266, was negative for D2 antigen. Staining various cell lines by D2 and other antiplasma cell antibodies, PCA-1 and CD38, showed that D2 is distinct from PCA-1 and CD38. The fresh myeloma cells of 14 myeloma patients were stained by D2 and for other plasma cell antigens. D2 strongly stained three samples obtained from patients with clinically aggressive myeloma, while CD38 stained all cases except one. PCA-1 was positive in nine samples and negative in five. PCA-1 expression was observed in plasma cells obtained from pleural effusion and peripheral blood, while PCA-1-negative cases were not found in such samples, suggesting that PCA-I expression was related to extramedullary invasion. The morphology of the myeloma cells, classified according to Greipp's criteria, showed that there was no correlation between plasma cell antigen expression and plasma cell morphology. Analysis of D2 antigen expression should provide more information about the heterogeneity of myeloma cells.
Polymorphism of tumour necrosis factor-alpha (TNF-α) at position −308 in relation to ankylosing spondylitis
Authors: VERJANS G M G M , BRINKMAN B M N , VAN DOORNIK C E M , KIJLSTRA A , VERWEIJ C L
Published: July 2010

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the localization, in the proximity of the HLA-B locus, and the biological activities of TNF-α, we investigated the association between AS and a single base polymorphism located at position – 308 of the TNF-α gene. An allele-specific polymerase chain reaction was developed to monitor this polymorphism. The frequency of the TNF-α alleles was determined in 66 AS patients and 37 healthy controls. The TNF-α allele frequency was not significantly different between AS patients and controls.
The demonstration of an essential role for macrophages in thein vivogeneration of IgG2a antibodies
Authors: BREWER J M , RICHMOND J , ALEXANDER J
Published: July 2010

BALB/c mice were depleted of macrophages by intravenous inoculation of dichloromethylene diphosphonate entrapped in liposomes 24 h before primary and 24 h before secondary sensitization intravenously with 100/μg bovine serum albumin (BSA) or ovalbumin (OVA). The effectiveness of macrophage depletion was confirmed by immunocytochemistry. Five days and 14 days after secondary challenge with BSA, plasma samples from these and control mice inoculated with empty liposomes were examined for the production of BSA-specific IgG1 and lgG2a antibodies. Macrophage depletion resulted in a significantly increased production of the Th2 lymphocyte-associated IgG1 isotype, while the production of specific IgG2a antibodies, produced under the influence of Th1 cells, was totally ablated. Similar results were obtained when OVA was used as the test antigen. Furthermore, analysis of interferon-gamma (IFN-γ) production after antigen or concanavalin A (Con A) restimulation in vitro indicated that macrophage depletion m vivo significantly reduced production of this Th1 cell-associated cytokine. These results provide strong in vivo and in vitro evidence for the macrophage being the antigen-presenting cell population responsible for Th1 cell activation.
Retroviruses and autoimmune rheumatic diseases
Authors: KALDEN J R , GAY S
Published: July 2010

In autoimmune rheumatic diseases, retroviruses have been repeatedly discussed as important etiologic factors. However, despite a considerable amount of indirect evidence that retroviruses might indeed be involved in triggering or perpetuating autoimmune rheumatic diseases, clear cut direct evidence is still missing. Studies on arthropathies associated with HIV-1 or HTLV-1 infection as well as new experimental animal models like the Tax transgene mice and new data from the MLRlpr mouse model might help to answer the questions how and by what mechanisms retroviral infection may lead to autoimmune rheumatic diseases. From data obtained in the MLR/lpr mouse it seems obvious that a potential link of retroviruses, apoptosis and autogenes to autoimmune diseases opens exciting new approaches to the study of rheumatic disease pathogenesis.
Mapping of B cell epitopes on steroid 17α-hydroxylase, an autoantigen in autoimmune polyglandular syndrome type I
Authors: PETERSON P , KROHN K J E
Published: July 2010

Earlier studies have shown that 17α-hydroxylase (P450cl7), steroid 21-hydroxylase (P45Dc21) and side-chain cleavage enzyme (P450scc) are the main autoantigens recognized by sera from patients with Addison's disease associated with autoimmune polyglandular syndrome type I (APS I). In this study we tried to identify the autoantigenic epitopes on P450c17 and compared the identified sequences with corresponding regions in two other adrenal autoantigens, P450scc and P450c21. A series of F450c17 cDNA fragments was expressed in Escherichia coli and the recognition of the corresponding protein fragments by patient sera was tested by immunoblotting. Four distinct epitope regions (ER) were found: ER1 (amino acids 122–148), ER2 (280–304), ER3 (396–432) and ER4 (466–508). B cell epitope prediction analysis showed that the four identified ERs were all located in regions of high predicted antigenicity. Homology search revealed that ER3 and ER4 but not ER1 and ER2 were related to similar sequences on P450c21. No significant homologies with P450SCC in these regions were found. Although all three P450 cytochromes are genuine autoantigens this finding suggests that the autoantibody reaction against P450c17 and P450c21 can partly be a result of immunological cross-reactivity.
Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells
Authors: BARANYI L , OKADA N , BARANJI K , TAKIZAWA H , OKADA H
Published: July 2010

Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 512 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 512 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymozan treatment abrogated the protective effect.
Soluble cell adhesion molecules and von Willebrand factor in children with Kawasaki disease
Authors: NASH M C , SHAH V , DILLON M J
Published: July 2010

Fifty-nine children with acute Kawasaki disease (KD), a childhood vasculitis, were compared with 35 children with fever due to infection and 48 healthy children. Levels of soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1)in the healthy children were double those found in adults. All three soluble cell adhesion molecules and von Willebrand factor (vWF) were higher in the children with KD than in the healthy children, but only sE-selectin, a marker for activated endothelial cells, and sICAM-1 were higher than in the febrile children. The high levels of vWF in KD appear to reflect the prominent acute-phase reaction. This information can help us to understand further the complex interactions between cytokines. circulating inflammatory cells and the vascular endothelium, and may lead to new therapeutic avenues in KD and other inflammatory diseases and vasculitides.
Large granular lymphocyte expansions in patients with Felty's syndrome: analysis using anti-T cell receptor Vβ-specific monoclonal antibodies
Authors: BOWMAN S J , BHAVNANI M , GEDDES G C , CORRIGALL V , BOYLSTON A W , PANAYI G S , LANCHBURY J S
Published: July 2010

Felty's syndrome (FS), the association of rheumatoid arthritis (RA) and idiopathic neutropenia, remains an unexplained phenomenon. HLA-DR4 is found in over 90% of cases. Patients with FS may have a T cell lymphocytosis of CD3+CD8+CD57+ large granular lymphocytes (LGL syndrome). In this study of 47 patients with FS, 19% had clear evidence for LGL expansions, while in total 42% had variable evidence for the LGL syndrome using currently available techniques. Of these T cell expansions, 76% were clonal, as demonstrated by Southern blotting and analysis with T cell receptor (TCR) β chain constant region probes. This technique may fail to detect clonal populations in some patients. Cytofluorographic analysis using antibodies specific for TCR β chains identified patients with clonal LGL expansions with results comparable to those obtained with Southern blotting. No evidence for shared Vβ usage among expansions from different patients was seen. The role of LGL in RA and FS is currently unclear, but this technique offers a practical and accessible means of identifying patients with LGL expansions, as a starting point for further investigation.
No evidence for an independent role of anti-heparan sulphate reactivity apart from anti-DNA in lupus nephritis
Authors: HYLKEMA M N , ZWET I V D , KRAMERS C , VAN BRUGGEN M C J , SWAAK A J G , BERDEN J H M , SMEENK R J T
Published: July 2010

The presence of anti-heparan sulphate (HS) reactivity in serum is closely related to the occurrence of nephritis in patients with systemic lupus erythematosus (SLE). Since patients with lupus nephritis in general also have high titres of anti-DNA antibodies, we wanted to clarify the relationship between anti-HS and anti-DNA reactivity in serum. Therefore, we studied longitudinally six patients with lupus nephritis who experienced 12 exacerbations of their disease, and five SLE patients without nephritis experiencing 10 periods of non-renal disease exacerbations. In addition, we tested single serum samples of another 24 patients obtained during a renal disease exacerbation and 22 sera of patients without nephritis. The sera of all patients were tested for anti-DNA (Farr assay) and anti-HS reactivity (ELISA). We confirmed that SLE patients during renal exacerbations have a significantly higher anti-HS reactivity than patients without nephritis (P < 0·003). In addition, patients with nephritis also had higher titres of anti-DNA antibodies during renal exacerbations than during non-renal exacerbations (P < 0·01). A correlation between anti-DNA and anti-HS reactivity was observed (r= 0·40, P < 0·02), which in itself explains the correlation between nephritis and anti-HS reactivity. Comparing sera from nephritis and non-nephritis patients matched for anti-DNA titre, we found no difference in anti-HS reactivity, and therefore must conclude that the anti-HS reactivity is a direct reflection of anti-DNA reactivity.
Defects in antigen-driven lymphocyte responses in common variable immunodeficiency (CVID) are due to a reduction in the number of antigen-specific CD4+ T cells
Authors: FUNAUCHI M , FARRANT J , MORENO C , WEBSTER A D B
Published: July 2010

T cells from patients with CVID have defects that may relate to the failure in vivo of B cell production of antibodies. Antigen-driven responses of T cells from CVID patients and normal subjects have been assessed by measuring DNA synthesis in vitro. Low density cells enriched for antigen-presenting dendritic cells were pulsed with purified protein derivative (PPD) and cultured with autologous T cells. Overall, T cells from CVID patients showed a significantly low mean response to PPD, although non-specific DNA synthesis induced in CVID T cells by IL-2 was within the normal range. However, mean PPD-specific T cell responses in CVID were not restored by IL-2 irrespective of the presence of monocytes. Depletion of CD8+ cells also failed to restore the mean PPD response of CVID CD4+ T cells. Limiting dilution analysis showed that in CVID there was a reduced frequency of antigen-specific cells within the T cell preparations. The mean frequency of the PPD-specific T cells in cultures from patients vaccinated with bacille Calmette-Guérin (BCG) was reduced to 1 in 109000 T cells compared with 1 in 18 600 T cells in BCG-vaccinated normal donors. These data show that the reduced PPD-specific response in CVID is due to a partial peripheral loss of antigen-specific cells.
Modulation of the IL-1 cytokine network in keratinocytes by intracellular IL-1α and IL-1 receptor antagonist
Authors: PHILLIPS W G , FELDMANN M , BREATHNACH S M , BRENNAN F M
Published: July 2010

The IL-1 cytokine network in epidermal cells was studied in vitro, using the spontaneously transformed HaCAT human keratinocyte line. Intracellular (ic) IL-1α and IL-1 receptor antagonist protein (IL-1Ra) following cell lysis were readily identified assayed using a capture ELISA; whereas in culture supernatants IL-1Ra was not detected, and IL-1α was present at only very low levels. Confluent cultures of HaCAT cells were shown to provide optimal conditions for the study, since confluence increased the icIL-1Ra: IL-1α ratio to a level as seen in vivo, which was independent of Ca2+ concentration in the culture medium. The IL-1Ra extracted from HaCAT cell lysates was functionally active, as demonstrated in the mouse thymocyte co-proliferation assay which could be blocked using a rabbit anti-IL-1Ra antibody. Transforming growth factor-beta (TGF-α) stimulated a dose-dependent increase in HaCAT cell 1L-1α without changing IL-1Ra concentration, with a resultant reduction in the iclL-1Ra: IL-1α ratio from 320:1 to 100:1. Similarly, TGF-α, interferon-gamma (IFN-γ), IL-6, and tumour necrosis factor-alpha (TNF-α) substantially increased HaCAT cell IL-1α, but had no effect on the IL-1Ra. with a concomitant reduction in the icIL-1Ra: IL-1α ratio. In contrast to their effects on monocytes, IL-4and IL-10 at biologically active levels had no effect on IL-1α, IL-1Ra or the icIL-1Ra: IL-1a ratio in confluent HaCAT cells. Hydrocortisone reduced IL-1α to below the limit of sensitivity of the ELISA, and induced a small increase in IL-1Ra of questionable biological significance. Thus, regulation of the IL-1 cytokine network in keratinocytes involves modulation of icIL 1α rather than of icIL-1Ra levels, and is markedly different from that noted in monocytes.
Chemokine expression in rheumatoid arthritis (RA): evidence of RANTES and macrophage inflammatory protein (MIP)-1β production by synovial T cells
Authors: Robinson E , Keystone E C , Schall T J , Gillett N , Fish E N
Published: July 2010

Earlier studies from this laboratory provided evidence for restricted cytokine expression in the T cell population in RA tissues. Specifically, IL-2, IL-4, IL-6 and interferon-gamma (IFN-γ) gene expression levels wore low. The selective chemoattractant and activation effects of chemokines on leucocytes identify them as potentially ideal candidates in mediating selective inflammatory processes in RA. Accordingly, we undertook studies to examine constitutive chemokine gene expression in RA tissues. RANTES, monocyte chemotactic protein-1 (MCP-1) and MIP-1β gene expression was examined in both the T and non-T cell populations in RA peripheral blood (PB), synovial fluid (SF) and synovial tissues (ST). Our results identified elevated levels of both RANTES and MIP-1β gene expression in circulating RA PB and SF T cells. By contrast, MCP-1 expression was virtually absent in RA PB, yet elevated MCP-1 mRNA levels were detected primarily in the non-T cell populations of the SF and ST samples. Histological examination of affected rheumatoid joints revealed extensive RANTES and MIP-1β expression in sites of lymphocyte infiltration and cell proliferation, namely the synovial lining and sublining layers. Fractionation of RA ST patient samples revealed that RANTES expression was restricted to the T cells, whereas MIP-1β expression was detected in both T and non-T fractions. These data suggest that MCP-1, MIP-1β and RANTES may have a central role in the trafficking of reactive molecules involved in immunoregulation and in the inflammatory processes in RA.
B cells capable of spontaneous IgG secretion in cerebrospinal fluid from patients with multiple sclerosis: dependency on local IL-6 production
Authors: Perez L , Alvarez-CermeÑO J C , Rodriguez C , RoldÁN E , Brieva J A
Published: July 2010

Cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients contains B cells capable of spontaneous IgG secretion in vitro. This study analyses the function and regulation of these cells. CSF cells obtained from nine MS patients actively produced IgG during 2–3 days in culture, and the activity decreased when CSF cells were cultured in serum-free medium. CSF cells from four controls did not secrete detectable IgG in vitro. Further experiments revealed that IL-6 played a role on MS CSF IgG-secreting cells, as can be deduced from the following findings: (i) the addition of exogenous IL-6, but not of other cytokines, to serum-free cultures restored missing CSF cell IgG secretion; (ii) the inclusion of anti-IL-6, but not of control, blocking MoAb reduced IgG secretion by CSF cells in fetal calf serum (FCS)-containing cultures; and (iii) CSF cells were capable of active IL-6 production in the presence of FCS. These results suggest that endogenous IL-6 production by MS CSF cells seems to be responsible for inducing CSF IgG-secreting B cells to reach terminal differentiation.
In vitroanalysis of metabolic predisposition to drug hypersensitivity reactions
Authors: Riley R J , Leeder J S
Published: July 2010

Idiosyncratic hypersensitivity reactions may account for up to 25% of all adverse drug reactions, and pose a constant problem to physicians because of their unpredictable nature, potentially fatal outcome and resemblance to other disease processes. Current understanding of how drug allergy arises is based largely on the hapten hypothesis: since most drugs are not chemically reactive per se, they must be activated metabolically to reactive species which may become immunogenic through interactions with cellular macromolecules. The role of drug metabolism is thus pivotal to the hapten hypothesis both in activation of the parent compound and detoxification of the reactive species. Although conjugation reactions may occasionally produce potential immunogens (for example, the generation of acylglucuronides from non-steroidal anti-inflammatory drugs such as diclofenac), bioactivation is catalysed most frequently by cytochrome P450 (P450) enzymes. The multifactorial nature of hypersensitivity reactions, particularly the role of often unidentified, reactive drug metabolites in antigen generation, has hampered the routine diagnosis of these disorders by classical immunological methods designed to detect circulating antibodies or sensitized T cells. Similarly, species differences in drug metabolism and immune system regulation have largely precluded the establishment of appropriate animal models with which to examine the immunopathological mechanisms of these toxicities. However, the combined use of in vitro toxicity assays incorporating human tissues and in vivo phenotyping (or, ultimately, in vitro genotyping) methods for drug detoxification pathways may provide the metabolic basis for hypersensitivity reactions to several drugs. This brief review highlights recent efforts to unravel the bases for hypersensitivity reactions to these therapeutic agents (which include anticonvulsants and sulphonamides) using drug metabolism and Immunochemical approaches. In particular, examples are provided which illustrate breakthroughs in the identification of the chemical nature of the reactive metabolites which become bound to cellular macromolecules, the enzyme systems responsible for their generation and (possibly) detoxification, and the target proteins implicated in the subsequent immune response.
Autoantibodies to histone, DNA and nucleosome antigens in canine systemic lupus erythematosus
Authors: Monestier M , Novick K E , Karam E T , Chabanne L , Monier J-C , Rigal D
Published: July 2010

Dogs can develop systemic lupus erythematosus syndromes that are clinically similar to those seen in humans. In contrast, previous observations suggest differences in their autoantibody reactivity patterns against histones and DNA which are components of the nucleosome in chromatin. The objective of this study was to assess comprehensively the levels of autoantibodies against histone, DNA and nucleosome antigens in a population of lupus dogs. The specificities of antibodies in lupus and control dog sera were determined using IgM- and IgG-specific reagents in an ELISA against a variety of chromatin antigens. When compared with control sera, IgG antibodies to individual histones H1, H2A, H3 and H4 were significantly higher in the lupus group. In contrast, we did not detect IgG antibodies specific for H2B, H2A-H2B, DNA, H2A-H2B-DNA or nucleosomes in lupus dogs. There was no significant increase in any of the IgM specificities tested. Therefore, the reactivity pattern to nucleosome antigens in canine lupus is restricted to IgG antibodies against individual histones H1, H2A, H3 and H4. This stands In contrast with human and murine lupus, where autoantibodies are directed against a wide variety of nucleosomal determinants, suggesting that unique mechanisms lead to the expansion of anti-histone antibody clones in canine lupus. The high incidence of glomerulonephritis in dog lupus suggests that anti-DNA antibodies are not required for the development of this complication, whereas IgG anti-histone antibodies may be relevant to its pathogenesis.
Analysis of γδ region usage in normal and diseased human intestinal biopsies and peripheral blood by polymerase chain reaction (PCR) and flow cytometry
Authors: Bucht A , SÖDerstrÖM K , Esin S , Grunewald J , Hagelberg S , Magnusson I , Wigzell H , GrÖNberg A , Kiessling R
Published: July 2010

The intestinal population of γδ T cell receptor (TCR)-bearing cells was characterized with regard to Vδ and Vγ subtype expression. For this purpose, we utilized V gene-specific PCR of mRNA prepared from intestinal biopsies. Predominant expression of the Vδ1 subtype was demonstrated in the small intestine of patients with coeliac disease and in the inflamed colon of patients with inflammatory bowel diseases (IBD: ulcerative colitis and Crohn's disease) as well as in colon biopsies taken from macroscopically normal areas of colon. Although intestinal γδ T cells preferentially expressed Vδ1, other Vδ transcripts could be detected, of which Vδ2 and Vδ5 were commonly expressed. Analysis of biopsies from mesenteric lymph nodes demonstrated a Vδ repertoire similar to the mucosa. In peripheral blood on the other hand, high expression of both Vδ2 and Vδ1 was found. The predominant expression of Vδ1 transcripts in the intestinal mucosa of IBD patients correlated well with protein cell surface expression as analysed by flow cytometry using Vδ1- and Vδ2-specific antibodies. Selective expansion of γδ T cells could not be demonstrated within the inflamed mucosa as shown by mRNA analysis and flow cytometry. Instead, IBD patients demonstrated a decreased proportion of TCR 76-carrying T cells in the inflamed mucosa compared with macroscopically normal area of colon. On the other hand, a significantly increased percentage of T cells bearing the γδ TCR was found in peripheral blood of patients with Crohn's disease compared with healthy individuals, indicating that local mucosal inflammation may influence the circulating γδ T cell population.
In vitroandin vivoT cell responses in mice during bronchopulmonary infection with mucoidPseudomonas aeruginosa
Authors: Stevenson M M , Kondratieva T K , Apt A S , Tam M F , Skamene E
Published: July 2010

In vitro and in vivo T cell responses were determined during the course of bronchopulmonary infection with mucoid Pseudomonas aeruginosa. T cell responses were compared in two inbred mouse strains, namely BALB/c mice, which are resistant to the establishment of chronic bronchopulmonary Ps. aeruginosa infection, and C57B1/6 mice, which have high numbers of bacteria in the lungs through 14 days post-infection. Unseparated lung cells and lung T cells from BALB/c mice exhibited significantly higher in vitro proliferative responses to both heat-killed Ps. aeruginosa and concanavalin A (Con A) than cells from C57B1/6 mice through 20 days post-intratracheal infection with 104 colony-forming units (CFU) Ps. aeruginosa. Proliferation of unseparated lung cells but not lung T cells from BALB/c mice infected 6 days previously with 105 CFU Ps. aeruginosa was suppressed in response to Con A; these cells were unresponsive to specific antigen. Suppression of lymphocyte proliferation in the lungs of C57B1/6 mice infected with 104 CFU Ps. aeruginosa and in BALB/c mice infected with 105 CFU was found to be mediated by adherent lung cells via the production of nitric oxide and prostaglandins. Determination of in vivo T cell-mediated responses in infected mice demonstrated that resistant BALB/c mice had high DTH and low Pseudomonas-specific antibody responses, while C57B1/6 mice had low DTH and high antibody levels, in particular, IgG2b and IgM.
Enhanced production of transforming growth factor-beta (TGF-β) during autologous mixed lymphocyte reaction of systemic sclerosis patients
Authors: OTA H , KUMAGAI S , MORINOBU A , YANAGIDA H , NAKAO K
Published: July 2010

Systemic sclerosis (SSc) is characterized by systemic fibrosis and microvascular lesions. As TGF-β is suggested to be related to skin fibrosis, we examined the production of TGF-β from peripheral mononuclear cells (MNC) of SSc patients. Since anti-TGF-β neutralizing antibody improved the defective proliferative response in autologous mixed lymphocyte reaction (AMLR) of SSc patients, TGF-β was thought to participate in the decreased AMLR of SSc patients. Greater amounts of TGF-β in the active as well as in the latent forms were produced during AMLR of SSc patients than that of normal subjects. It was suggested that TGF-β excessively produced from the MNC of SSc patients might play a major role in the fibrosis of the patients during AMLR-like in vivo responses.
Selective recruitment of lymphocyte subsets to the inflamed appendix
Authors: SOO K S , MICHIE C A , BAKER S R , WYLLIE J H , BEVERLEY P C L
Published: July 2010

Total lymphocyte counts and the distribution of lymphocyte subsets were determined in peripheral venous blood and appendiceal mononuclear cells from 60 patients who underwent appendicectomy for the clinical diagnosis of appendicitis. A significant peripheral lymphopenia was observed in the 46 patients with histologically confirmed acute appendicitis which was accompanied by an increase in the appendiceal lymphocyte concentration. There was an even greater depletion of CD45RO+ (memory) T lymphocytes in peripheral blood and an increase in the inflamed appendix. Reciprocal changes were observed in the CD45RA+ (naive) T lymphocyte subset. These changes were reflected in the local arterial and venous CD45RA and CD45RO T lymphocyte subsets. Proliferation studies showed an expanded functional repertoire of T lymphocytes in the inflamed appendix. Selective recruitment of memory T lymphocytes from the peripheral blood to the inflamed appendix was demonstrated.
Cell-mediated autoimmunity in patients with Wegener's granulomatosis (WG)
Authors: BALLIEUX B E P B , VAN DER BURG S H , HAGEN E C , VAN DER WOUDE F J , MELIEF C J M , DAHA M R
Published: July 2010

Despite the well described infiltration of cells of the cellular immune system in vasculitic lesions and the granuloma formation in patients with WG, the role of T cell-mediated autoimmunity in WG is not clear. Reports of T cell proliferation in response to neutrophil azurophilic granule proteins are contradictory. In this study we have assessed the proliferation of T cells of WG patients to purified proteinase 3 (PR3) and to total azurophilic granule proteins in two different assays. In addition to the classical proliferation assay with isolated peripheral blood mononuclear cells, we have used a whole blood proliferation assay. In both assays we found proliferative responses to PR3 in patients with WG. The number of patients reacting to the azurophilic granule extract was higher than the patients reacting to the purified PR3, suggesting that other autoantigens may also be involved. We have identified epitopes of PR3 that may be potential targets of class I-restricted T cell responses in the context of HLA-A*0201, the most common MHC class I molecule. These epitopes were determined by the binding of synthetic PR3 peptides to HLA-A*0201 on the antigen-processing defective cell line, T2. In addition, T cell lines were established from tissue biopsies, obtained from WG patients, and assessed for cytolytic reactivity against T2 cells, preloaded with synthetic PR3 peptides. We conclude that T lymphocytes of WG patients have increased proliferative responses to purified PR3 and to a larger extent to non-fractionated proteins of azurophilic granules of polymorphonuclear neutrophilic leucocytes (PMN).
Autoantibodies against eukaryotic protein L7 in patients suffering from systemic lupus erythematosus and progressive systemic sclerosis: frequency and correlation with clinical, serological and genetic parameters
Authors: NEU E , VON MIKECZ A H , HEMMERICH P H , PETER H-H , FRICKE M , DEICHER H , GENTH E , KRAWINKEL U
Published: July 2010

Recent studies have shown that sera of patients suffering from systemic autoimmune diseases contain autoantibodies directed against the eukaryotic ribosomal protein L7 [1]. In the present study we screened a large panel of sera from patients with systemic lupus erythematosus (SLE) for the presence of anti-L7 autoantibodies and their relationship to clinical, serological and genetic parameters of SLE. By means of an ELISA employing recombinant protein L7 as antigen we detected anti-L7 autoantibodies in 172 of 506 SLE sera (34%). Negative correlations were observed between the presence of anti-L7 autoantibodies, serum IgG levels and proteinuria; a potentially positive relationship existed with lung fibrosis. In order to analyse further this possibility we screened sera of 129 patients suffering from progressive systemic sclerosis (PSS) for anti-L7 reactivity; 45 of these patients had lung fibrosis. Of the PSS patients, 41% exhibited anti-L7 autoantibodies, but positive reactions were evenly distributed among patients with and without lung fibrosis. Protein L7 thus represents a major autoantigen of systemic autoimmune diseases, but does not so far define a distinct subpopulation of patients.