bluuebunny/crossref_metadata_2025 huggingface dataset:
Failure of IgG production due to a defect in the opening of the chromatin structure of Iγ1 region in a patient with IgG and IgA deficiency
Authors: Kondo N , Inoue R , Kasahara K , Kaneko H , Kameyama T , Orii T
Published: July 2010

Patients with common variable immunodeficiency (CVID) display reduced levels of two or all three of the major immunoglobulin isotypes, and the deficiency is characterized by failure of B cells to differentiate into plasma cells in many cases. A patient (14 years old, female) showed normal serum IgM levels and low serum IgG and IgA levels, including low levels of all IgG subclasses. Northern blot analysis suggested that the patient's B cells may be defective at the immunoglobulin heavy chain isotype switch. The germ-line Cγ1 transcript was amplified from cDNA of healthy controls by the addition of recombinant IL-2 (rIL-2) to pokeweed mitogen-stimulated peripheral mononuclear cells or Staphylococcus aureus Cowan I (SAC)-stimulated IgM-producing lymphoblastoid cell lines (LCL) transformed by Epstein-Barr virus, while it was not amplified from cDNA of the patient. In the Iγ1 region of LCL cultured with SAC plus rIL-2, the inner cytosine in the 5′C-C-G-G 3′sequence nearest the 3′site of the Iγ1 region, at least, was not completely unmethylated in the patient. Moreover, the DNase I hypersensitive site was not induced in the patient's LCL by SAC plus rIL-2. These results indicate that the defects of the immunoglobulin heavy chain isotype switch in the patient's B cells are due to failure in the synthesis of germ-line Cγ transcripts, and this may be caused by defects in opening of the chromatin structures of specific switch regions.
A role for IL-4 in immunologically mediated enteropathy
Authors: Mowat A MCI , Widmer M B
Published: July 2010

A number of clinical enteropathies are associated with a local cell-mediated immune (CMI) response, and experimental evidence indicates that cytokines are responsible for the intestinal pathology. We show here that depletion of IL-4 using MoAb or a soluble form of the IL-4 receptor (IL-4R) prevents the jejunal manifestations of a proliferative form of murine graft-versus-host reaction (GVHR) characterized by crypt hyperplasia and recruitment of intraepithelial lymphocytes (IEL). Depletion of IL-4 did not prevent the appearance of villus atrophy in a destructive model of GVHR, and had no effect on any indices of systemic immunity. These results indicate that IL-4 may play a selective role in mediating proliferative aspects of intestinal immunopathology, and suggest that this cytokine may provide a useful target for immunotherapy.
Interferon-gamma (IFN-γ) production by human T lymphocytes uponLegionella pneumophilastimulationin vitro
Authors: Kitsukawa K , Nakamoto A , Koito H , Matsuda Y , Saito A , Yamamoto H
Published: July 2010

In vitro immune responses to Legionella pneumophila were investigated. When human peripheral blood lymphocytes (PBL) from healthy volunteers were stimulated with formalin-killed L. pneumophila for 7 days in vitro, strong proliferative responses were observed. The responding cells were shown to be a CD4a T cell subset. It was also found that the CD4 T cells secreted significant amounts of IFN-γ into the PBL culture supernatant. The production of IFN-γ and IL-4 by PBL was measured semiquantitatively by reverse transcriptase-assisted polymerase chain reaction (RT-PCR) methods. Formalin-killed or live L. pneumophila- stimulated PBL expressed the mRNA for IFN-γ but not the mRNA for IL-4. The results suggest that the whole bacterium, as opposed to the supernatant, predominantly stimulates Th1 type helper T cells. The cloned T cells specific for L. pneumophila expressed the mRNA for IFN-γ but not for IL-4. In contrast to formalin-killed or live L. pneumophila stimulation, when PBL were stimulated with the bacterial culture supernatant, the proliferating T cells produced the mRNA for IL-4 as well as for IFN-γ. A significant correlation between the proliferative response to formalin-killed L. pneumophila and IFN-γ release in culture was observed (r= 0.6932, P 0.001) in PBL from 30 healthy volunteers. From these in vitro studies, it is suggested that the whole L. pneumophila bacterium and their soluble antigens stimulate T cells in a manner which results in a different pattern of cytokine production.
IL-8 release from cultured peripheral blood monocytes of patients with glomerulonephritis
Authors: Matsumoto K
Published: July 2010

IL-8 is a chemotactic cytokine with proinflammatory and growth-promoting activities. The release of IL-8 was measured in supernatants of cultured peripheral blood monocytes (PBM) that were obtained from patients with glomerulonephritis (GN) and healthy controls. Spontaneous and lipopoly-saccharide (LPS)-induced IL-8 release was significantly higher in PBM isolated from patients with IgA nephropathy (IgAN) and membranous nephropathy (MN) compared with normal controls. These results raise the question of whether IL-8 contributes to the ongoing pathogenesis of GN, We cannot relate IL-8 release to clinical and laboratory parameters in IgAN and MN patients. Thus, disease progression in vivo may not be accompanied by increased or sustained IL-8 release.
Longer local retention of adoptively transferred T-LAK cells correlates with lesser adhesion molecule expression than NK-LAK cells
Authors: YAMAMOTO T , YONEDA K , OSAKI T , YOSHIMURA N , AKAGI N
Published: July 2010

The local retention of adoptively transferred lymphokine (IL-2)-activated killer (LAK) cells was examined in 11 patients with head and neck carcinoma. Unseparated lymphocytes, T and natural killer (NK) cells isolated from patients were cultured with IL-2 for 7 days, labelled with 99mTc-HMPAO, and immediately injected back into the respective donors via the superficial temporal artery or locally into the tumour tissue. The injected LAK cells were periodically traced using a gamma camera, and the LAK cell retention rate was calculated from the radioactivity. One hour after the injection, about 70% of the locally infiltrated LAK cells remained in the tumour tissue, while about half of the LAK cells transferred via the regional artery were dislodged from the tissue. LAK cells induced from T cells (T-LAK) were retained in the tissue for a longer time than LAK cells induced from NK cells (NK-LAK). T-LAK were less chemotactic and less adherent to human umbilical vein endothelial cells (EC), and showed lesser migration through EC. Flow cytometric analysis revealed higher expression of CD11a, CD11b, CD18 and CD49d on NK-LAK compared with T-LAK. MoAbs against these adhesion molecules suppressed adhesion and migration of LAK cells. These results indicate that the rapid disappearance of NK-LAK from the tissue is associated with their greater chemotactic and adhesive as well as migratory activities depending on differing expression of adhesion molecules.
Effect of postponed treatment with an anti-tumour necrosis factor (TNF) F(ab')2 fragment on endotoxin-induced cytokine and neutrophil responses in chimpanzees
Authors: VAN DER POLL T , LEVI M , CATE H TEN , JANSEN J , BIEMOND B J , HAAGMANS B L , EERENBERG A , VAN DEVENTER S J H , HACK C E , CATE J W TEN
Published: July 2010

TNF is considered to be an intermediate factor in endotoxin-induced release of other cytokines and endotoxin-induced neutrophil degranulation. Little is known about the effect of postponed treatment with anti-TNF in primate endotoxin models. To assess the effect of delayed treatment with anti-TNF in endotoxaemia, six healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg). In three of these animals the administration of endotoxin was followed after 30 min by a bolus i.v. injection of the anti-TNF F(ab')2 fragment MAK 195F (0.1 mg/kg). Post-treatment with MAK 195F completely prevented the appearance of TNF activity in serum elicited by endotoxin, and markedly reduced the rises in the serum concentrations of IL-6 and IL-8. In addition, the endotoxin-induced increases in the type I and type II soluble TNF receptors were also profoundly inhibited by MAK 195F, suggesting that TNF is involved in the release of its own soluble receptors in endotoxaemia, Neutrophilic leucocytosis was not affected by MAK 195F. In contrast, MAK 195F did significantly abrogate neutrophil degranulation, as measured by the plasma concentrations of lactoferrin. These results indicate that treatment with anti-TNF 30 min after the administration of endotoxin is still effective in attenuating the induction of the cytokine network and of neutrophil degranulation.
Vaccination of patients deficient in a late complement component with tetravalent meningococcal capsular polysaccharide vaccine
Authors: PLATONOV A E , BELOBORODOV V B , PAVLOVA L I , VERSHININA I V , KÄYHTY H
Published: July 2010

Eighteen patients with late complement component deficiency (LCCD) were immunized with meningococcal capsular polysaccharide vaccine. The LCCD patients had experienced one-to-five meningococcal infections before vaccination, but their immunological and clinical status was normal at the time of immunization. Serum samples from vaccinated complement-sufficient relatives of the LCCD patients and healthy Russian male adults were used as controls. Total and immunoglobulin-specific concentrations of antibodies to group A, C, W135, and Y capsular polysaccharides were determined by enzyme immunoassay in serum samples taken before and 1–108 weeks after immunization. The individual preimmunization and post-immunization antibody concentrations varied greatly. The median antibody concentrations of the LCCD patients increased significantly after vaccination, and were not significantly different from those of the control groups. The antibody concentrations remained elevated for at least 1 year after vaccination. The post-immunization antibody concentrations correlated with the number of meningococcal infections within 10 years before vaccination. In spite of the vaccination two LCCD patients experienced a meningococcal disease 9 and 12 months, respectively, after vaccination.
The kinetics and distribution of C9 and SC5b-9in vivo: effects of complement activation
Authors: GREENSTEIN J D , PEAKE P W , CHARLESWORTH J A
Published: July 2010

Many diseases associated with complement activation are characterized by tissue deposition of components of the terminal complement complex (TCC). The ninth component of complement (C9) plays an important role in the cytolytic effects, and may contribute to the non-lethal cell-regulating functions of the TCC [1]. In this study we examined the behaviour of radiolabelled human C9 and its soluble complexed form SC5b-9 in vivo in order to determine the effects of complement activation on its turnover, distribution and molecular size. In normal rabbits the metabolic parameters of 125I-C9 (median and range) were: plasma half-life (t1/2) 25.9 (20.6-29.5) h, fractional catabolic rate (FCR) 5.7 (5.3-7.0)%/h, and extravascular/intravascular ratio (EV/IV) 0.7 (0.6-1.1). The distribution of radiolabelled C9 amongst body tissues was similar to that observed for rabbit serum albumin (RSA). Activation of the complement cascade with i.v. injection of cobra venom factor (CVF) resulted in rapid disappearance of C9 from the plasma and accumulation of protein-bound radiolabel in the spleen (exceeding the plasma concentration) and the liver. RSA metabolism and distribution were unaffected by CVF. Fine performance liquid chromatography (FPLC) gel filtration of plasma samples suggested that monomeric C9 was the only major radiolabelled protein present during normal turnovers, whereas CVF administration was accompanied by the prompt appearance of a high mol. wt species consistent in size with SC5b-9. When injected directly, 125I-SC5b-9 disappeared rapidly from the plasma, falling by 50% in 0.7 (0.6-0.8) h, and less than 15% remaining after 4 h with accumulation of protein-bound label in the spleen and liver. These results demonstrate the complexity of C9 metabolism during complement activation.
Dendritic cells in synovial fluid of chronic inflammatory arthritis lack CD80 surface expression
Authors: SUMMERS K L , DANIEL P B , O'DONNELL J L , HART D N J
Published: July 2010

Dendritic cells (DC) act as potent primary antigen-presenting cells in many immune responses and therefore may have a role in the initiation and perpetuation of the synovial inflammation in chronic inflammatory arthritis. To examine their function, it is important to isolate fresh DC from arthritic joints without aberrant activation. We have developed a technique using minimal cell manipulation to isolate DC from the synovial fluid of chronic arthritic patients. Using this method, DC were shown to be potent allostimulatory cells, with 63–90% of cells lacking lineage-specific markers (lin-), but positive for MHC class II molecules. Two morphologically distinct populations of these cells were identified in 10 out of 13 DC preparations. Both populations expressed CD40, intercellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3 and leucocyte function associated antigen-3 (LFA-3), but the predominant population, which was larger and more typical of cultured blood DC, had a higher density of these antigens compared with the minor population, which were smaller and morphologically similar to lymphocytes. Two new MoAbs which label activated human blood DC, HB15 (CD83) and CMRF-44, were tested. CD83 labelled very weakly or not at all, whereas CMRF-44 was positive on the larger cells only. Likewise, the costimulator molecule, B7/BB1 (CD80), was not detected on the surface of either synovial lin- cell population, reverse transcriptase polymerase chain reaction (RT-PCR) showed little or no CD80 mRNA, and no binding of the CTLA-4Ig fusion protein was found. These results suggest that synovial DC are not, despite the inflammatory environment, in a fully activated state.
The rheumatoid arthritis-associated autoantibodies to filaggrin label the fibrous matrix of the cornified cells but not the profilaggrin-containing keratohyalin granules in human epidermis
Authors: SIMON M , VINCENT C , HAFTEK M , GIRBAL E , SEBBAG M , GOMÈS-DAUDRIX V , SERRE G
Published: July 2010

Since they were first described, serum IgG antibodies to the stratum corneum of rat oesophagus epithelium, highly specific for rheumatoid arthritis (RA), have been consensually called anti-keratin antibodies (AKA). However, we recently demonstrated that they actually recognize three new proteins of rat oesophagus epithelium distinct from cytokeratins, and also human epidermal filaggrin. In this work we provided further evidence that AKA and RA-associated anti-filaggrin autoantibodies are the same antibodies. Moreover, analysing by indirect immunofluorescence on human skin a large series of 212 well characterized RA sera and anti-filaggrin autoantibodies purified from RA sera by affinity chromatography. we demonstrated the specific binding of AKA to the stratum corneum of human epidermis and the absence of any staining of the granular keratinocytes. This binding was confirmed and the AKA antigen precisely localized in human epidermis by immunoelectron microscopy. The antigen was found to be restricted to the filaggrin-containing intracellular fibrous matrix of the corneocytes, up to the desquamating cells. In contrast, MoAbs directed to human filaggrin and to profilaggrin, its precursor, not only stained the intracellular matrix of the tower corneocytes but also the keratohyatin granules of the granular cells, where profilaggrin is stored. These results reinforced by the absence of immunoblotting reactivity of RA sera to profilaggrin suggest that the epitopes recognized by AKA are absent from profilaggrin. Their identification may provide more insight into the pathogenesis of RA.
Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT)
Authors: KARUSSIS D M , A-KARUSSIS U VOURK , LEHMANN D , ABRAMSKY O , BEN-NUN A , SLAVIN S
Published: July 2010

MRL-lpr/lpr mice spontaneously develop a severe autoimmune syndrome, characterized by massive generalized lymphadenopathy, arthritis, arteritis, dermatitis and immune complex-mediated glomerulonephritis. Bone marrow transplantation (BMT) from MHC-matched systemic lupus erythematosus (SLE)-resistant donors to susceptible recipients has proved effective in correcting autoimmune manifestations in autoimmune-prone mice. We investigated the effect of syngeneic BMT from MRL/lpr (donor) to immunocompromised MRL/lpr (recipient), after purging the bone marrow inoculum with MoAbs against mature T cells (anti-Thy 1.2). All the untreated mice developed lymphadenopathy and by the age of 36 weeks five of the eight were dead; in contrast, all the mice which underwent syngeneic BMT following acute immunosuppression with total body irradiation (900 cGy) (TBI) remained disease-free. In an additional experiment, it was found that conditioning with cyclophosphamide (CY) before BMT was more effective than TBI in inhibiting delayed-onset autoimmune manifestations (mean survival 350 days in the CY group and 305 days in the TBI group, versus 197 days in untreated controls). Under both immunosuppressive regimens T cell-depleted bone marrow grafts produced far better results than did unmanipulated BMT. Following syngeneic BMT the incidence of proteinuria and the level of serum anti-DNA (dd) antibodies were significantly reduced, compared with that of the age-matched untreated controls. CY was more effective than TBI in reducing the anti-DNA titres. Likewise. T depletion of bone marrow inocula before BMT induced a more drastic drop in autoantibodies, following both CY and TBI conditioning protocols. After syngeneic BMT (either CY or TBI) no signs of lymphadenopathy were observed even at an advanced age. Upon histopathological examination, the BMT-treated mice displayed normal glomeruli with occasional minimal signs of glomerulonephritis. Syngeneic T cell-depleted BMT following acute cytoreduction of anti-self immune lymphocytes may represent a new therapeutic approach for drug-resistant autoimmune diseases.
Anti-inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation
Authors: SEKUT L , YARNALL D , STIMPSON S A , NOEL L S , BATEMAN-FITE R , CLARK R L , BRACKEEN M F , MENIUS J A , CONNOLLY K M
Published: July 2010

Inhibitors of cyclic nucleotide phosphodiesterases are known to suppress lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-α) production in vitro in human monocytes. The most potent of these have selectivity for type IV PDEs, suggesting that this class of PDE is the major type involved in the regulation of human TNF-α production. Using compounds of two distinct chemical structural classes, a quinazolinedione (CP-77 059) and a 4 arylpyrrolidinone (rolipram). we show here that PDE-IV-specific inhibitors are also potent in suppressing LPS-induced TNF-α production in vitro in sodium periodate-elicited murine macrophages (IC50s of 1 and 33, respectively). We then report the in vivo anti-inflammatory effect of PDE-IV inhibition in five murine models of inflammation: (i) elevation of serum TNF-α induced by a subtethal LPS injection; (ii) LPS-induced endotoxic shock; (iii) LPS/galactosamine-induced endotoxic shock; (iv) carrageenan-induced paw oedema; and (v) adjuvant arthritis. Following a sublethal (5 μg/mouse) injection of LPS, serum TNF-α levels in mice peaked sharply, reaching concentrations of 3–12 ng/ ml 90 min after injection. In this sublethal LPS assay, CP-77 059 was about 30 times more potent than rolipram, with a minimum effective dose of 0.1 mg/kg versus 3 mg/kg for rolipram. This rank order is in keeping with the relative in vitro IC50S for CP-77059 and rolipram, as well as their relative Ki against the human PDE-IV enzyme (46 nM and 220 nM, respectively). In LPS-induced endotoxic shock, rolipram and CP-77 059 at relatively high doses of 30 and 10 mg/kg, respectively, significantly reduced serum TNF-α levels, and also inhibited mortality 66%. In the LPS/galactosamine shock model, in which mice are rendered exquisitely sensitive to LPS by co-injection with galactosamine, only 0.1 μg of LPS/mouse Is necessary for serum TNF-α elevation and death. Both rolipram and the CP-77059 caused dose-dependent reduction of serum TNF-α and lethality. In the carrageenan-induced paw oedema model, in which there is a pronounced local TNF-α response (without a serum TNF-α elevation), rolipram significantly inhibited paw swelling as well as localized TNF-α levels in the paw. In the adjuvant arthritis model, a chronic model of inflammation also possessing localized TNF-α elevation in the inflamed paw, rolipram and CP-77059 suppressed ankle swelling and radiological evidence of joint damage. These data are consistent with a major role for PDE-IV in regulation of TNF-α production and inflammatory responses in murine systems. It suggests a potential therapeutic use for PDE-IV-specific inhibitors in inflammatory disease such as rheumatoid arthritis, septic shock and other inflammatory diseases where TNF-α has been postulated to be a contributing factor in the pathology of the disease.
Tyrosine phosphorylation pathway is involved in interferon-gamma (IFN-γ) production; effect of sodium ortho vanadate
Authors: GIOVANNETTI A , AIUTI A , PIZZOLI P M , PIERDOMINICI M , AGOSTINI E , OLIVA A , DIANZANI F , AIUTI F , PANDOLFI F
Published: July 2010

The molecular mechanisms regulating IFN-γ production have yet to be well characterized. We describe here how treatment of activated cultures of peripheral blood mononuclear cells (PBMC) with the phosphotyrosine phosphatases (PTP) inhibitor sodium ortho vanadate results in greatly enhanced IFN-γ production. Conversely, cellular proliferation of the same cultures is profoundly inhibited by treatment with vanadate, while the expression of IL-2R and DR molecules on activated lymphocytes remains substantially unmodified. Increased IFN-γ production, but not inhibition of cellular proliferation, was also observed in mitogen-activated vanadate-treated Jurkat cells. On the other hand, IFN-γ production induced in cultures of PBMC treated or not with vanadate, was strongly inhibited by incubation with the protein tyrosine kinase (PTK) inhibitor herbimycin A. As a result of the inhibited phosphatase activity, substrates for PTK become hyperphosphorylated on tyrosine residues, as shown by Western blot analysis of cell lysates from cultures of PBMC treated with vanadate. We suggest that the tyrosine phosphorylation pathway plays a role in regulating IFN-γ production.
α1-Antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis
Authors: SAVIGE J A , CHANG L , COOK L , BURDON J , DASKALAKIS M , DOERYf J
Published: July 2010

α1-antitrypsin (α1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of α1-AT phenotypes associated with dysfunctional α1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of α1-AT. Phenotypes usually associated with a moderate or severe reduction in α1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n= 2), S (n= 1) or Z (n= 1) (P < 0·05). However, the corresponding α1-AT serum levels were normal (n= 3) or elevated (n= 1). None of the 31 sera with anti-PR3 antibodies had low levels of α1-AT. No abnormal α1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of α1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum oi-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal α1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal α1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have α1-AT phenotypes that are usually associated with low serum levels of α1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal α1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal α1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal α1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.
Analysis of T cell responses to the autoantigen in Goodpasture's disease
Authors: DERRY C J , ROSS C N , LOMBARDI G , MASON P D , REES A J , LECHLER R I , PUSEY C D
Published: July 2010

Goodpasture's disease is a rare form of glomerulonephritis characterized by the production of autoantibodies to the glomerular basement membrane (GBM). In order to understand the development of autoimmunity to the GBM, it is important to examine mechanisms underlying T cell responses to the autoantigen. A MoAb PI, with the same specificity as patients’ autoantibodies, was used to affinity-purify the antigen from collagenase-digested human GBM. This material was enriched in the NCI domain of the α3 chain of type IV collagen (α3(IV)NC1), known to be the principal target of anti-GBM antibodies, but also contained lower quantities of α4(IV)NC1. In proliferation assays, T cells from 11/14 patients with Goodpasture's disease showed significant responses (SI ± 2·0) to affinity-purified human GBM. Peak responses were demonstrated at 7 or 10 days at antigen concentrations of 10–30 μg/ml. As in other autoimmune disorders, the presence of autoantigen-reactive T cells was also demonstrated in 5/10 healthy volunteers. Tissue typing revealed that all patients possessed HLA-DR2 and/or -DR4 alleles, while normal individuals whose T cells responded possessed DR2 and/or DR7 alleles. The specificity of the T cell response in Goodpasture's disease was further investigated using monomeric components of human GBM purified by gel filtration and reverse phase high performance liquid chromatography (HPLC). Two antigenic monomer pools were obtained, which were shown by amino-terminal sequence analysis to contain α3(IV)NC1 and α4(IV)NC1, respectively. In all patients tested, significant T cell proliferation was observed in response to one or both of these α(IV)NC1 domains. These results demonstrate that patients with Goodpasture's disease possess T cells reactive with autoantigens known to be recognized by anti-GBM antibodies.
Stimulation of the expression and the enzyme activity of aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 on human renal cell carcinoma cells and renal tubular epithelial cells by T cell-derived cytokines, such as IL-4 and IL-13
Authors: RIEMANN D , KEHLEN A , LANGNER J
Published: July 2010

Aminopeptidase N (APN) and dipeptidylpeptidase TV (DPIV) are transmembrane type II molecules widely distributed in mammalian tissues. In recent years, the interest in cell surface peptidases has increased considerably because, among other things, several reports indicate roles of ectopeptidases in tumour cell metastasis. Investigations into the regulation of APN and DPIV on tumour cells are rare. We report, for the first time, that IL-4 and IL-13 can up-regulate protein expression as well as enzymatic activity of both the peptidases on renal carcinoma cells and renal tubular epithelial cells in culture. The analysis of mRNA by competitive polymerase chain reaction (PCR) confirmed our results with respect to the APN increase at the level of gene expression. IL-1β and tumour necrosis factor-alpha (TNF-a) augmented the IL-4-induced effect with respect to APN but not to DPIV, A 5-day incubation with interferon-gamma (IFN-γ) increased protein expression, especially of APN and, to a lesser extent, also of DPIV, whereas no significant increase in enzymatic activity could be observed. Small concentrations of transforming growth factor-beta I (TGF-β1) inhibit the expression and enzyme activity of DPIV. IL-6, IL-7. IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any effect on APN and DPIV. For a prospective therapeutic regimen with T cell-derived cytokines it has to be considered that—besides their effect on tumour cell growth—cytokines might affect surface ectopeptidases involved in tumour cell adhesion processes. The inhibition of APN and DPIV could be a new approach to suppression of cancer spread.
Sequence analysis of the germ-line VH gene corresponding to a nephritogenic antibody in MRL/lpr lupus mice
Authors: ONO M , YAMAMOTO T , KYOGOKU M , NOSE M
Published: July 2010

In order to investigate the genetic origin of nephritogenic antibodies in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice, we isolated the germ-line heavy chain variable region (VH) gene corresponding to the nephritogenic antibody, B1, derived from an unmanipulated MRL/lpr mouse. Injection of this antibody into C.B-17/Icr-scid/scid mice resulted in the generation of wire loop-like glomerular lesions resembling those of lupus nephritis. Nucleotide sequences of this germ-line VH gene showed no replacement mutation in the VH region of the B1 antibody. Furthermore, this gene was identical to that found in the C3H/HeJ-lpr/lpr strain of mice. Our results suggest that germ-line VH genes can encode nephritogenic antibodies without somatic mutation, even in a mouse strain not prone to lupus.
Evidence for a CD14+ population of monocytes in inflammatory bowel disease mucosa—implications for pathogenesis
Authors: GRIMM M C , PAVLI P , VAN DE POL E , DOE W F
Published: July 2010

Lipopolysaccharide (LPS) is abundant in the intestinal lumen. CD14 is the receptor for the LPS-LPS binding protein complex, and its presence on mononuclear phagocytes allows cell activation by pg/ml concentrations of LPS. We have shown that the recently recruited blood monocyte in inflammatory bowel disease mucosa is CD14+. This study examined the expression of CD14 on macrophages in inflamed (n= 13) and uninflamed (n= 7) intestine by immunohistochemistry, and on disaggregated lamina propria mononuclear cells (12 from inflamed, 17 from uninflamed intestine) and peripheral blood mononuclear cells (n= 26) by flow cytometry, using a panel of three MoAbs directed against CD14. Immunohistochemistry revealed that 3·7% of macrophages in uninflamed intestine were CD14+, while 25·1 % of macrophages in active inflammatory bowel disease expressed CD14 (P < 0·02). Flow cytometry demonstrated that CD14 expression by macrophages from Crohn's disease and ulcerative colitis was augmented significantly (P= 0·02 and P= 0·01, respectively) compared with uninflamed intestine, with a discrete population of macrophages in inflammatory bowel disease, not present in normal intestine, which strongly expressed CD14. The characteristically high levels of CD14 on blood monocytes were unaffected by the presence of intestinal inflammation. Given the exposure of lamina propria cells to LPS present in the lúmen of the terminal ileum and colon, the increased numbers of CD14+ macrophages in inflammatory bowel disease may result in greatly increased production of inflammatory mediators, thereby suggesting a mechanism for the perpetuation of mucosal inflammation.
Evaluation of soluble IL-6 receptor concentration in serum and epithelial lining fluid from patients with interstitial lung diseases
Authors: YOKOYAMA A , KOHNO N , HIRASAWA Y , KONDO K , ABE M , INOUE Y , FUJIOKA S , FUJINO S , ISHIDA S , HIWADA K
Published: July 2010

We measured soluble IL-6 receptor (sIL-6R) levels in serum and bronchoalveolar lavage fluids (BALF) from patients with interstitial pneumonia of unknown etiology (IP) (n= 17), sarcoidosis (n= 8) and normal control subjects (n= 10), to investigate its role in pulmonary diseases. Soluble IL-6R was determined by an ELISA. The volume of epithelial lining fluid (ELF) in BALF was estimated using an urea method. We found that levels of sIL-6R in serum, BALF, and ELF from patients with IP or sarcoidosis were significantly higher than those from normal subjects. Furthermore, levels of sIL-6R in BALF or ELF were significantly correlated with those of albumin, indicating that sIL-6R, together with albumin, may enter ELF as a result of the increased permeability caused by pulmonary inflammation. Thus most of the sIL-6R in ELF would be from serum, and relatively small amounts of it might be produced locally. However, sIL-6R levels in ELF, but neither serum nor BALF, were significantly correlated with levels of C-reactive protein in patients with IP. These results suggest that both systemic and local production of sIL-6R are increased, and raised sIL-6R is involved in the modulation of systemic and local inflammatory responses in patients with IP and sarcoidosis.
Immunodeficiency in RFM/(T6xRFM)F1 mouse chimaeras with lethal host-versus-graft syndrome
Authors: HARD R C , MONTOUR J L , FUCHS B A
Published: July 2010

Rather than central tolerance, the perinatal inoculation of related F1 hybrid spleen cells into inbred mice may result in host-versus-graft (HVG) reactions manifested as transient autoimmunity, or as a lethal immunodeficiency syndrome. RFM/(T6xRFM)F1 chimaeras with lethal disease die in 30 days with lymphosplenomegaly, immune complexes and impaired immune responses. The present studies used in vitro proliferation assays to show that the HVG reaction caused hyperplasia sufficient to account for the lymphosplenomegaly, while also causing severe impairment of splenic and nodal cell responses to concanavalin A (Con A) and to bacterial lipopolysaccharide (LPS). By 25 days, HVG mice could not distinguish between self and non-self as judged by mixed lymphocyte reactions (MLR) to RFM, (T6xRFM)F1 and third party A/J cells. There were no indications that host cells reactive to F1 donor cells had undergone clonal deletion, anergy or expansion. Flow cytometry revealed that donor T lymphocytes achieved stable engraftment, mostly in the nodes, despite the HVG reaction. Taken together with previous observations, these studies showed that HVG reactions in young parent F1/chimaeras can result in an immunodeficiency state which is characterized by an early appearing, profound and persistent impairment of both host and donor T and B cell functions. The results suggest that HVG reactions can contribute directly to immune deficits seen after clinical allogeneic bone marrow transplantation.
An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes
Authors: PHIPPS D J , RHED-DOOB P , MACFADDEN D K , PIOVESAN J P , MILLS G B , BRANCHE D R
Published: July 2010

Following infection with HIV, patients exhibit lymphocyte dysfunction before the loss of CD4+ T cells. The major HIV surface glycoprotein, gp120, can modulate lymphocyte function in vitro; however, the mechanism by which gp120 affects T lymphocyte signal transduction is controversial. We have used Peptide T, a synthetic octapeptide derived from a conserved, CD4 binding region of gp120, to examine gp120-related modulation of lymphocyte signal transduction. Activation of lymphocytes through the T cell receptor (TCR) in collaboration with cell surface accessory molecules results in rapid increases in tyrosine phosphorylation, probably through the recruitment and activaiion of src-family protein tyrosine kinases (PTK) sueh as lck and fyn which have been implicated in mediating the proximal signalling events mediated through the TCR. To identify potential mechanisms by which gp120 could modulate the function of T lymphocytes, we determined the effect of Peptide T on normal, activated peripheral blood lymphoblasts. Treatment of normal, activated peripheral blood lymphoblasts with Peptide T (10-90m) for 60 min transiently reduced levels of protein tyrosine phosphorylation (ptyr). Reduction in levels of cellular ptyr was associated with transient inhibition of the activity of total cellular and CD4-associated p56lck kinase activity (80%). Peptide T also induced a small delayed reduction in the p59fyn activity (up to 42%). Despite the decrease in total cellular ptyr levels, pp60c–src kinase activity was increased 11-fold following treatment with Peptide T. Peptide T pretreatment also induced tyrosine phosphorylation of a 48-kD CD4-associated protein, indicating that Peptide T may have multiple effects. Peptide T did not alter the levels of total cellular p56lck enzyme, nor did it directly inhibit the activity of purified p56lck. These results are consistent with a Peptide T-dependent modulation of PTK regulation, and support the potential of gp120 to interfere with T lymphocyte signal transduction in activated T lymphocytes.
HIV-specific lymphoproliferative responses in asymptomatic HIV-infected individuals
Authors: PONTESILLI O , CARLESIMO M , VARANI A R , FERRARA R , GUERRA E C , BERNARDI M L , RICCI G , MAZZONE A M , D'OFFIZI G , AIUTI F
Published: July 2010

In vitro lymphoproliferative responses to HIV-1 recombinant antigens (gp160, p24, and Rev protein) were studied in 83 patients with asymptomatic HIV-1 infection (CDC groups 11 and III) and circulating CD4 lymphocyte numbers > 400/mm3. Significant response to at least one of the three antigens was detected in 52.4% of the subjects, but the responses were weak, and concordance of the response to the three antigens was rare, the frequency of individuals responding to each antigen not exceeding 22.4%. Increasing frequencies of response were observed when recall antigens (tetanus toxoid and Candida albicans glycomannoprotein) (65.5%) and anti-CD3 MoAb (76.6%) were used as stimuli. Although a significant association between lymphocyte response to p24, but not gp160, and steadiness of CD4 lymphocyte numbers before the assay was observed, no predictive value for lack of CD4 cell decrease was confirmed for either antigen, and fluctuation of the responses to HIV antigens was seen during subsequent follow up. The panel of T cell assays used could be regarded as appropriate for monitoring both HIV-specific responses and T lymphocyte function during immunotherapy with soluble HIV antigens.
The expression of IL-2, IL-4 and interferon-gamma (IFN-γ) mRNA using liver biopsies at different phases of acute exacerbation of chronic hepatitis B
Authors: Fukuda R , Ishimura N , Nguyen TX , Chowdhury A , Ishihara S , Kohge N , Akagi S , Watanabe M , Fukumoto S
Published: July 2010

To investigate the hypothesis that Th1 phenotype cytokines are associated with the increasing activity of hepatitis and Th2 phenotype cytokines with decreasing activity in the liver of chronic viral hepatitis, expressions of the mRNA of the cytokines IL-2, IFN-γ and IL-4 in the liver of 23 patients with chronic hepatitis B were investigated by reverse transcription polymerase chain reaction. Patients were divided into three groups according to the phase of acute exacerbation of hepatitis as increasing (n= 9), decreasing (n= 8), and stable phase (n= 6). Both IL-2 and IFN-γ mRNA were preferentially expressed in increasing phase than in decreasing phase (P < 0.01, P < 0.05, respectively) and associated with the high serum alanine aminotransferase (ALT) level. On the other hand, IL-4 mRNA was detected in decreasing phase with significant frequency compared with increasing phase (P < 0.05). However, expression of IL-4 mRNA was not associated with serum ALT level. Our results suggest that Th1 phenotype cytokines up-regulate and Th2 phenotype cytokines down-regulate the liver inflammation of chronic viral hepatitis.
Expression of a unique protein on colon cancer cells that reacts with a novel monoclonal antibody and ulcerative colitis serum
Authors: HASSAN T , KANISAWA Y , MEYERS S , DASGUPTA A , DAS K M
Published: July 2010

We earlier developed a MoAb, 7E12H12 (IgM isotype), against a protein present in normal colonic epithelial cells. To examine if 7E12H12-reactive protein is expressed in colon cancer cells and is recognized by ulcerative colitis (UC)-associated autoantibody, we investigated several colon cancer cell lines. 7E12H12 reactivity against the cells was examined by indirect immunofluorescence assay and whole cell ELISA against six colon cancer cell lines HT-29, LoVo, COLO 205, DLD-1, LS 180 and SW 1116. A competitive ELISA was developed using 7E12H12 MoAb and patients' serum to examine the cross-reactive antibodies in the serum. Among the six colon cancer cell lines only LS 180, DLD-l and SW 1116 reacted with 7E12H12 MoAb, while others did not. The mean (s.e.m.) inhibition of the binding or 7E12H12 MoAb to LS 180 cells by UC serum (n= 51) was 42±2±1%, whereas in normal subjects (n= 17) it was 14±2±6%, in Crohn's disease (n= 19) it was 15±3±2±5%, in infectious diarrhoea (n= 10) it was 11%±3%, and in systemic lupus erythematosus (n= 10) it was 2%±0±6%. The inhibition by the UC group was significantly (P<0±001 - <0±000l) higher than any of the non-UC groups, and this inhibition was mainly by IgG1 antibody. The protein in the specific colon cancer cells recognized by the 7E12H12 MoAb cross-reacts with UC-IgG1 antibody and may provide an in vitro system to examine the autoimmune mechanisms in UC.
Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy
Authors: ALLEN A C , HARPER S J , FEEHALLY J
Published: July 2010

The mechanism of IgA deposition in the kidneys in IgA nephropathy is unknown, Mesangial IgA is of the IgA I subclass, and since no consistent antigenic target for the IgA I has been described, we have investigated the glycosylation of the molecule, as a potential non-immunological abnormality which may contribute to its deposition. IgA 1 is rich in carbohydrate, carrying N-linked moieties in common with IgG, but also O-linked sugars, which are rare in serum proteins, and not expressed by IgG or lgA2, Lectin binding assays were designed to examine the expression of terminal galactose on the N-linked carbohydrate chains of purified serum IgG and IgAI, and the O-linked sugars of IgAI and C1 inhibitor (one of the very few other serum proteins with O-linked glycosylation). No evidence was found for abnormalities of N-linked glycosylation of either isotype in IgA nephropathy compared with matched controls. However, in IgA nephropathy, reduced terminal galactosylation of the hinge region O-linked moieties was demonstrated; this was not seen in C1 inhibitor, which showed normal or increased galactosylation of the O-linked sugars. This abnormality of IgA1 has considerable implications for the pathogenesis of IgA nephropathy, since the O-linked sugars lie in an important functional location within the IgA1 molecule, close to the ligand of Fc receptors. Changes in the carbohydrates in this site may therefore affect interactions with receptors and extracellular proteins, leading to anomalous handling of the IgA1 protein in this condition, including failure of normal clearance mechanisms, and mesangial deposition.
Autoantibody response to the Ro/La particle may predict outcome in neonatal lupus erythematosus
Authors: SILVERMAN E D , BUYON J , LAXER R M , HAMILTON R , BINI P , CHU J-L , ELKON K B
Published: July 2010

This study was undertaken to determine the role of antibodies against both recombinant Ro (r-Ro) and La (r-La) proteins and polypeptides derived from the recombinant La protein in predicting fetal and neonatal outcome in children at risk to develop neonatal lupus erythematosus (NLE). All sera were obtained in the perinatal period and quantitative ELISA assays were used. We collected 41 maternal sera within 2 months of delivery of a child with NLE (21 with congenital heart disease block (CHB) and 20 with dermatologic NLE) and 19 sera from anti-Ro and/or anti-La antibody-positive mothers with systemic lupus erythematosus (SLE) who delivered a child without NLE. All sera were tested for anti-r-La and anti-r-Ro antibodies by ELISA, and most sera were tested for antibodies directed against La polypeptides by immunoblot. We found significantly higher anti-r-La antibody levels in the sera from mothers of children with NLE compared with sera from mothers of unaffected children (0.67 ± 0.43 versus 0.14 ± 0.30; P < 0.0001). There was a statistically significant difference in the mean anti-r-La levels between the sera of mothers of children with CHB compared with dermatologic NLE (0.51 ± 0.45 versus 0.83 ± 0.37 respectively; P= 0±0091). When we examined antibodies directed against the recombinant 52-kD Ro protein, there was a statistically significant elevation of titres in the sera of mothers of NLE children (0.77 ± 0.35) compared with non-NLE mothers (0.29 ± 0.39; P<0.0001). There was no difference in the r-Ro levels between mothers of children with dermatologic NLE compared with CHB (0.82 ± 0.37 versus 0.71 ± 074; P= 0.32). When we examined polypeptides derived from the recombinant La protein, the mean number of polypeptides recognized by sera from mothers of children with NLE was significantly higher than the mean number of polypeptides recognized by sera from mothers of unaffected children (5.1 ± 0.54 versus 2.3 ± 0.54 respectively; P < 0.0001). More importantly, when we examined the individual polypeptides. we found that only sera from mothers of children with NLE and not from mothers of unaffected children recognized a polypeptide designated DD (30%versus 0%, respectively). These studies indicate that the autoantibody response to the Ro/La particle can differentiate sera from mothers of children with NLE and sera from mothers of unaffected children. Furthermore, there was a difference In the anti-La autoantibody response between mothers of children with CHB and dermatologic NLE. Antibodies directed against the DD polypeptide derived from the recombinant La protein were seen only in the sera of mothers of children with NLE.
γδ T cell receptor gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies
Authors: O'HANLON T P , MESSERSMITH W A , DALAKAS M C , PLOTZ P H , MILLER F W
Published: July 2010

Autoreactive αβ T cells have been implicated as playing a primary pathogenic role in a group of diseases characterized by chronic muscle inflammation known as the idiopathic inflammatory myopathies (IIM). γδ T cells, a distinct and enigmatic class of T cells, play a less certain role in a variety of human autoimmune diseases including the IIM. In an attempt to understand the significance of γδ T cells in the IIM, we utilized a sensitive polymerase chain reaction (PCR) technique to evaluate γδ T cell receptor (TCR) gene expression in 45 muscle biopsies obtained from 42 IIM patients (17 polymyositis, 12 dermatomyositis, and 13 inclusion body myositis). γδ TCR gene expression was not detected in 36 specimens, the majority of muscle biopsies surveyed. γδ TCR gene expression by muscle-infiltrating lymphocytes was detected among nine clinically heterogeneous patients. We further analysed the junctional sequence composition of the Vγ3 and Vδ1 transcripts, whose expression was prominent among γδ positive patients. DNA sequence analysis of Vγ3 amplification products from two patients revealed the presence of several productively rearranged transcripts with amino acid sequence similarities within the Vγ3-N-Jγ junctional domain. No amino acid sequence similarities were evident within the Vδ-N-Dδ-N-Jδ region of Vδ1 transcripts amplified from four patients, although a distinct and dominant clonotype was detected from each patient. Our cumulative data suggest that unlike αβ T cells, γδ T cells do not play a prominent pathologic role in the IIM. In fact, the sporadic nature of γδ TCR gene expression detected among these patients implies that γδ T cell infiltration, when it occurs, is a secondary event perhaps resulting from non-specific inflammatory processes.