bluuebunny/crossref_metadata_2025 huggingface dataset:
Human thymic epithelial cells maintain long‐term survival of clonogenic myeloid and erythroid progenitor cells in vitro
Authors: Takenaka Katsuto , Harada Mine , Fujisaki Tomoaki , Nagafuji Koji , Mizuno Shinichi , Miyamoto Toshihiro , Otsuka Teruhisa , Gondo Hisashi , Okamura Takashi , Niho Yoshiyuki
Published: July 2010

Precursor cells that migrate into the thymus are still multipotent. Therefore, thymic epithelial cells (TECs) may provide microenvironments not only for T‐cell development, but also for maintenance of multipotent precursor cells until they undergo T‐cell commitment. In the present study, we performed long‐term cultures of CD34+ bone‐marrow (BM) cells on TEC lines that were derived from cortical epithelial cells of post‐natal thymus, to investigate whether human TECs could maintain long‐term non‐lymphoid haematopoiesis. Haematopoietic cells maintained in direct contact with established TEC lines were able to generate clonogenic progeny to both myeloid and erythroid cells for periods in excess of 5 weeks. Their abilities to support colony‐forming units of granulocytes–macrophages (CFU‐GM) and burst‐forming units of erythroids (BFU‐E) were almost equal to those of BM stromal cells. We observed similar results by using cloned TEC lines derived by limiting dilution, as well as those by using parental TEC lines. Colony‐forming activities were maintained even when haematopoietic progenitor cells were physically separated from TEC lines and cultured on microporous membrane. These observations indicate that haematopoiesis maintained in TEC‐contact long‐term cultures may depend on soluble factors produced by TEC lines. Our results suggest that thymic cortical epithelial cells have the ability to support not only the differentiation of haematopoietic cells, but also long‐term survival of clonogenic myeloid/erythroid progenitor cells.
Cytotoxic T‐lymphocyte precursor frequency (CTLp‐f) as a tool for distinguishing permissible from non‐permissible class I mismatches in T‐cell‐depleted allogeneic bone marrow transplantation
Authors: Van Der Meer Arnold , Joosten Irma , Schattenberg Anton V. , De Witte Theo J. M. , Allebes Wil A.
Published: July 2010

Matching for HLA has been the gold standard in bone marrow donor selection. But, with the ever increasing number of identified HLA alleles, it is becoming more difficult to find a fully HLA‐identical donor other than a sibling. Retrospective analysis revealed that HLA mismatches do not necessarily give rise to acute graft‐versus‐host‐disease (GVHD). However, we have no means of defining these ‘permissible’ mismatches before bone marrow transplantation (BMT). Thus, we set out to establish whether functional matching by means of helper and cytotoxic T‐lymphocyte precursor frequency analysis (HTLp‐f and CTLp‐f respectively) can be applied to this end. Fifty‐five recipient–donor pairs other than HLA‐identical siblings, the recipient of which received a T‐cell‐depleted graft, were analysed by high‐resolution HLA typing and/or HTLp‐f/CTLp‐f analysis. The predictive value of the CTLp‐f assay for development of acute GVHD was confirmed. More importantly, our data indicate that the CTLp‐f assay was able to discriminate permissible from non‐permissible HLA‐A, ‐B or ‐Cw mismatches, but not for DRB/DQB mismatches. The absolute number of alloreactive CTLs present in the graft correlated with the risk of acute GVHD. Although HTLp‐f and CTLp‐f together had a high negative predictive value, HTLp‐f outcome by itself was not correlated with acute GVHD. As we have no evidence yet that HTLp‐f or CTLp‐f can identify permissible DRB/DQB mismatches, high‐resolution matching for these antigens remains the best option. The combination of high‐resolution DRB/DQB typing and the CTLp‐f assay would enable the accurate prediction of the risk of acute GVHD while extending the pool of potential donors. Furthermore, it would enable adjustment of the number of T‐ cells in the graft accordingly to improve clinical outcome.
Neutrophil‐specific reduction in the expression of granulocyte–macrophage colony‐stimulating factor receptor subunits in myelodysplastic syndromes
Authors: Shikama Yayoi , Shichishima Tsutomu , Ohto Hitoshi , Jubinsky Paul T. ,   Yukio. Maruyama
Published: July 2010

The proliferative and differentiative response of neutrophils to granulocyte–macrophage colony‐stimulating factor (GM‐CSF) is known to be impaired in patients with myelodysplastic syndromes (MDS). To investigate the mechanisms of the defective response in MDS, we examined expression levels of GM‐CSF receptor α (GMRα) and common β (βc) subunits on CD16+ neutrophils, CD14+ monocytes and CD3+ T cells from 26 MDS patients and 10 healthy controls using flow cytometry. Expression of GMRα was significantly decreased on the neutrophils of five out of 26 patients and was not specific for any FAB subtype. In contrast, βc expression on neutrophils was significantly reduced in 14 out of 26 patients with a higher proportion occurring in the advanced stages of MDS including refractory anaemia with excess of blasts (RAEB), RAEB in transformation (RAEBt) and overt leukaemia compared with refractory anaemia (RA)/RA with ringed sideroblasts (RARS) or healthy controls. Decreased βc also correlated with the degree of hypogranular neutrophil morphology and increased infection. Expression of both subunits on T cells and monocytes in MDS was similar to normal controls. Polymerase chain reaction amplification of reverse‐transcribed mRNA isolated from the affected neutrophils suggests that the reduction of βc may result from decreased message levels. The observed reduction in GM‐CSF receptor expression could account for the impaired proliferative and maturational responses in MDS.
Generation of functionally mature dendritic cells from the multipotential stem cell line FDCP‐mix
Authors: Schroeder Timm , Lange Claudia , Strehl John , Just Ursula
Published: July 2010

Dendritic cells (DCs) are crucial components of the immune system because of their unique ability as antigen‐presenting cells for the initiation of a primary immune response. DCs, macrophages (Ms) and granulocytes (Gs) are believed to originate from a common myeloid progenitor cell. However, little is known about the molecular mechanisms leading to DC sublineage commitment. To establish a cell system that allows the molecular and biochemical analysis of DC differentiation and activation, we used the murine non‐leukaemic, multipotential stem cell line FDCP‐mix. FDCP‐mix cells were cultured in various amounts of GM‐colony stimulating factor (CSF) and interleukin (IL)‐4 for up to 16 d and analysed for morphology, expression of CD34, c‐kit, Gr‐1, Mac‐1, CD40, MHC‐I, MHC‐II and co‐stimulatory molecules (CD80, CD86) using flow cytometry, and for their capacity to present foreign antigen to autologous T cells. Up to d 7, the majority of FDCP‐mix cells consisted of cells differentiating along the G and M lineage. Thereafter, the number of dendritic cells increased until d 13. Differentiation along the DC lineage vs. the G and M lineage was favoured when FDCP‐mix cells were cultured in high concentration GM‐CSF (500 U/ml) throughout the culture and IL‐4 from d 9 onwards. The dendritic cells generated from FDCP‐mix cells were large, non‐adherent cells with veiled processes and expressed MHC II, CD40, CD80 and CD86. After pulsing with a foreign antigen (keyhole limpet haemocyanin), FDCP‐mix‐derived dendritic cells stimulated [3H]‐thymidine incorporation of naive T‐cells in an autologous mixed lymphocyte reaction (MLR). Our results show that functionally mature dendritic cells are generated from the multipotential stem cell line FDCP‐mix. This cell line thus provides the unique possibility of establishing multipotential transgenic cell lines capable of differentiation along the DC lineage. The experimental system described here should prove a valuable tool for studying DC differentiation and function.
Stem cell transplantation for the treatment of Fanconi anaemia using a fludarabine‐based cytoreductive regimen and T‐cell‐depleted related HLA‐mismatched peripheral blood stem cell grafts
Authors: Boulad Farid , Gillio Alfred , Small Trudy N. , George Diane , Prasad Vinod , Torok‐Castanza Joanne , Regan Anne D. , Collins Nancy , Auerbach Arleen D. , Kernan Nancy A. , O'reilly Richard J.
Published: July 2010

We have employed a new cytoreductive regimen to transplant two patients with Fanconi anaemia (FA), using T cell‐depleted two HLA‐allele disparate related peripheral blood stem cell transplants (PBSCTs). Patient 1, a 5‐year‐old male with FA and aplastic anaemia, initially received an HLA two‐antigen mismatched unrelated cord blood transplant and failed to engraft. He received fludarabine (Flu) and cyclophosphamide (Cy), followed by a CD34+ E‐rosette− (CD34+E−), T cell‐depleted, granulocyte colony‐stimulating factor (G‐CSF)‐mobilized PBSCT from his HLA B‐DRB1 mismatched father. He received anti‐thymocyte globulin (ATG), steroids, FK506 and G‐CSF after transplant for rejection and graft‐versus‐host disease (GVHD) prophylaxis. The patient is now 23 months after SCT with no evidence of GVHD and with full haematopoietic and immune reconstitution. Patient 2, a 10‐year‐old boy with FA and myelodysplastic syndrome, received single‐dose total body irradiation (SDTBI), Flu and Cy followed by a CD34+E−, T‐cell‐depleted, G‐CSF‐mobilized PBSCT from his HLA B‐DRB1 mismatched sister. He also received ATG, steroids, FK506 and G‐CSF after transplant. The patient is now 12 months after SCT in complete remission with no evidence of GVHD. Absolute neutrophil counts (ANC) of > 1 × 109/l were achieved on day 11 and day 10 post transplant respectively. Both patients are fully engrafted. In summary, we report two successful T‐cell‐depleted stem cell transplants from mismatched related donors for the treatment of Fanconi anaemia, using a fludarabine‐based cytoreduction. Both patients experienced minimal toxicity, rapid engraftment and no GVHD.
Procalcitonin in paediatric cancer patients: its diagnostic relevance is superior to that of C‐reactive protein, interleukin 6, interleukin 8, soluble interleukin 2 receptor and soluble tumour necrosis factor receptor II
Authors: Fleischhack Gudrun , Kambeck Isabell , Cipic Dubravka , Hasan Carola , Bode Udo
Published: July 2010

Sensitive parameters of inflammation are rare in neutropenic cancer patients. In this study, procalcitonin (PCT), C‐reactive protein (CRP), interleukin 6 (IL‐6), IL‐8, the soluble IL‐2 receptor (sIL‐2R) and the soluble tumour necrosis factor receptor II (sTNFRII) were evaluated for their diagnostic relevance in febrile episodes of cancer patients. Plasma or serum levels of these parameters were determined in neutropenic children with febrile episodes (n = 122) classified according to both the kind of infection [60 cases of fever of unknown origin (FUO), 28 cases of localized infection, 13 cases of pneumonia, 20 cases of bacteraemia, one case of fungaemia] and the World Health Organization (WHO) score of chemotherapy‐induced mucositis. At baseline and during the febrile episodes, the highest levels of all parameters were observed in cases of gram‐negative bacteraemia. However, in FUO and localized infections, low or only slightly elevated median levels of all parameters were documented. The degree of chemotherapy‐induced mucositis did not influence the value of any parameter. In comparison with the other inflammatory parameters, PCT (optimum cut‐off level 0·5 μg/l) was a more sensitive and more specific parameter in the diagnosis of high‐risk (gram‐negative bacteraemia) and low‐risk (FUO) episodes, as well as in the sequential assessment of all febrile neutropenic episodes.
Are aberrant BCR–ABL transcripts more common than previously thought?
Authors: Wilson G. A. , Vandenberghe E. A. , Pollitt R. C. , Rees D. C. , Goodeve A. C. , Peake I. R. , Reilly J. T.
Published: July 2010

We report the use of multiplex polymerase chain reaction (PCR), using 4% polyacrylamide gel electrophoresis (PAGE) for the detection of BCR‐ABL transcripts in Philadelphia‐positive disease. Three out of 50 cases [two out of 37 chronic myeloid leukaemia (CML), one out of 13 acute lymphoblastic leukaemia (ALL)] possessed rare breakpoints; an e19a2 and e13a3 in CML and an e1a3 in the ALL. We suggest that multiplex PCR using 4% PAGE and optimized for smaller transcript detection may lead to a higher detection rate of rare BCR‐ABL breakpoints. Multiplex PCR, however, failed to distinguish e13a2 from e1a3 transcripts. Finally, the presence of e13a3 in CML supports the view that abl exon 2 sequences are unnecessary for the pathogenesis of ‘classic’ CML.
PNH cells are as sensitive to T‐cell‐mediated lysis as their normal counterparts: implications for the pathogenesis of paroxysmal nocturnal haemoglobinuria
Authors: Karadimitris Anastasios , Notaro Rosario , Koehne Gunther , Roberts Irene A. G. , Luzzatto Lucio
Published: July 2010

The mechanism responsible for the bone marrow failure that is almost invariable in paroxysmal nocturnal haemoglobinuria (PNH) is unknown. Based on the close association between PNH and idiopathic aplastic anaemia, a plausible pathogenetic model predicts that, in PNH, autoreactive T cells specific for haemopoietic stem cells (HSCs) cause depletion of normal HSCs, whereas PNH HSCs escape this T‐cell‐mediated attack. In this study, we addressed the hypothesis that PNH HSCs are resistant to the cytotoxic effect of T cells because they lack surface expression of one or more glycosylphosphatidylinositol (GPI)‐linked molecules. We tested the sensitivity of normal and PNH Epstein–Barr virus (EBV)‐transformed B‐cell lymphoblastoid cell lines (BLCLs) to the cytotoxic effect of autologous EBV‐specific T‐cell lines and clones from a patient with PNH in an in vitro experimental system. We found that the PNH BLCLs were no less sensitive to T‐cell‐mediated cytotoxicity than non‐PNH isogenic BLCLs, indicating that GPI‐linked molecules on the surface of target cells are not required for killing by T cells. This suggests that the mechanism whereby PNH HSCs survive T‐cell attack is not because of the lack of surface expression of one or more GPI‐linked molecules. By implication, other mechanisms become more probable.
Pralatrexate, a novel class of antifol with high affinity for the reduced folate carrier‐type 1, produces marked complete and durable remissions in a diversity of chemotherapy refractory cases of T‐cell lymphoma
Authors: O'Connor Owen A. , Hamlin Paul A. , Portlock Carol , Moskowitz Craig H. , Noy Ariela , Straus David J. , MacGregor‐Cortelli Barbara , Neylon Ellen , Sarasohn Debra , Dumetrescu Otila , Mould Diane R. , Fleischer Martin , Zelenetz Andrew D. , Sirotnak Frank , Horwitz Steven
Published: July 2010

T‐cell lymphomas (TCLs) are characterised by poor responses to therapy with brief durations of remissions. An early phase study of pralatrexate has demonstrated dramatic activity in patients with relapsed/refractory disease. Of the first 20 lymphoma patients treated, 16 had B‐cell lymphoma and four had refractory aggressive TCL. All four patients with TCL achieved a complete remission. Patients with B‐cell lymphoma achieved stable disease at best. For each TCL patient, the response was more durable than their best response with chemotherapy. This early experience is the first to document this unique activity of pralatrexate in TCL.
The histone deacetylase inhibitor, PXD101, potentiates bortezomib‐induced anti‐multiple myeloma effect by induction of oxidative stress and DNA damage
Authors: Feng Rentian , Oton Ana , Mapara Markus Y. , Anderson Gülsüm , Belani Chandra , Lentzsch Suzanne
Published: July 2010

Clinical trials have shown the high anti‐myeloma activity of the proteasome inhibitor bortezomib. The present study examined the activity of bortezomib combined with PXD101, a histone deacetylase inhibitor, against multiple myeloma (MM) and osteoclastogenesis. Treatment of myeloma cell lines with combinations of bortezomib and PXD101 led to synergistic inhibition of proliferation and induction of cell death. The combination significantly decreased the viability of primary human CD138+ myeloma cells but not of bone marrow mononuclear cells. Further studies showed a dose‐dependent activation of caspases‐3, ‐8 and ‐9 and nuclear fragmentation in myeloma cells. Bortezomib/PXD101 treatment markedly triggered reactive oxygen species (ROS) generation that was accompanied by p53, H2A.X and p38–mitogen‐activated protein kinase phosphorylation. ROS generation could be blocked by the free radical scavenger N‐acetyl‐l‐cysteine. The combination of bortezomib and PXD101 also resulted in synergistic inhibition of osteoclast formation. In conclusion, bortezomib and PXD101 have different molecular targets. The combination induces cell death in myeloma cells via ROS‐mediated DNA damage and also inhibits osteoclastogenesis. Therefore, this study provides the rationale for the clinical evaluation of bortezomib combined with PXD101 in patients with MM.
Platelet glycoprotein VI‐related clinical defects
Authors: Arthur Jane F. , Dunkley Scott , Andrews Robert K.
Published: July 2010

Human patients with defects associated with the platelet collagen receptor, glycoprotein (GP)VI, are rare and usually described as having a mild bleeding disorder. However, here we review clinical profiles of patients with familial or acquired GPVI defects, revealing the bleeding defect is often severe and associated with immune dysfunction. GPVI is a member of the immunoreceptor family, and co‐expressed on platelets with Fc receptor γ‐chain (FcRγ). Ligand binding to GPVI leads to activation of platelet integrins, in particular αIIbβ3 that mediates platelet aggregation; and activation of endogenous platelet metalloproteinases resulting in ectodomain shedding and release of a soluble GPVI fragment. Increasing evidence supports the functional importance of GPVI/FcRγ in thrombus formation at arterial shear rates, and expression levels of platelet GPVI may be a marker of thrombotic risk. Over the past 20 years, patients have been reported with GPVI‐related defects involving: (i) an acquired deficiency, resulting from (a) anti‐GPVI autoantibodies or (b) other causes; or (ii) a congenital deficiency, where (c) GPVI is not expressed or (d) is expressed in a dysfunctional form with defective signalling to αIIbβ3. Clinical consequences of GPVI‐related defects may be uniquely informative about the role of platelet GPVI in health and disease.
Basophil activation test in the diagnosis of sulphite‐induced immediate urticaria
Authors: García‐Ortega P. , Scorza E. , Teniente A.
Published: July 2010

Cite this as: P. García‐Ortega, E. Scorza and A. Teniente, Clinical & Experimental Allergy, 2010 (40) 688–690.
Polyamine oxidase activity in rheumatoid arthritis synovial fluid
Authors: FERRANTE A , STORER R J , CLELAND L J
Published: July 2010

Oxidation of polyamides by polyamine oxidases (PAO) leads to the generation of highly reactive aminoaldehydes which have been shown to have a variety of effects, including killing of pathogenic microorganisms and regulation of leucocyte functions. Data presented here show that PAO are present in synovial fluid from patients with rheumatoid arthritis. This finding may have important implications in the various properties attributed to synovial fluid which includes anti-inflammatory activity
Macrophage heterogeneity in human fetal tissue. Fetal macrophages
Authors: OLIVER A M
Published: July 2010

The immunophenotype of the macrophage population in human fetal tissue was studied, using a panel of monoclonal antibodies against cells of the macrophage/monocyte lineage. Using a double-labelling technique two main populations were observed in tissue from 14 weeks of estimated gcstational age (EGA): EBM11 + DR + and EBM11 DR− cells of which a small proportion were also RFD7+. Most macrophages were negative with 3.9, an antibody specific for the adhesion molecule P150.95 and LP9 which is specific for a lysosomal enzyme. The exception to this was a small population of positive cells in the thymus. Small numbers of 3.9+ cells were also infrequently observed in tissue at and above 17 weeks of EGA. while occasional RFD9+ cells were only observed in most tissues, before this time. The higher percentage of macrophages were DR+ DQ−DP−. with a few DQ+ cells appearing at 15 weeks of EGA. In the thymus. DQ+ cells outnumbered DP+ cells especially in the medulla. These results indicate the heterogeneous and immature nature of the fetal macrophage population and point to the importance of age, tissue-specific factors and probable immune mediators in macrophage differentiation.
Decreased serum levels of IgE and IgE-binding factors in individuals infected with HTLV-I
Authors: MATSUMOTO T , MIIKE T , MIZOGUCHI K , YAMAGUCHI K , TAKATSUKI K , HOSODA M , KAWABE T , YODOI J
Published: July 2010

The cell surface expression of low-affinity Fc epsilon receptor (FceRII), which is identical to CD23, and the serum levels of IgE binding factors (IgE BFs), a soluble form of FceRII, and IgE were investigated in HTLV-I-infected individuals, and the results were compared with those for HTLV-I-negative controls. The occurrence of FcεRII+ mononuclear cells did not differ among the blood samples from the HTLV-I-infected groups; however, the levels of serum IgE BFs were significantly decreased (P<0.001) in patients with adult T cell leukaemia (ATL), compared with HTLV-I-negative controls. The serum IgE levels were also significantly decreased in these groups, including cases of ATL (P<0.001) and HTLV-I-associated myelopathy (P<0.05), and HTLV-I carriers (p<0.05), as compared with HTLV-I-negative controls. Since cases of strongyloidiasis or filariasis are frequently found among HTLV-I-infected individuals in Japan, these results may explain in part their defective defence mechanisms for parasites. The possible involvement of IgE BFs in the development of immune deficiency state in HTLV-I infection is also discussed.
In vitroproduction of tumour necrosis factor and prostaglandin E2 by peripheral blood mononuclear cells from tuberculosis patients
Authors: CADRANEL J , PHILIPPE C , PEREZ J , MILLERON B , AKOUN G , ARDAILLOU R , BAUD L
Published: July 2010

We investigated the production of tumour necrosis factor-alpha (TNF-α) and prostaglandin E2 (PGE2) by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy controls. PBMC from tuberculosis patients generated constitutively more TNF-α than did control PBMC. This production was significantly higher for patients with high-grade fever and cachexia. The increase of TNF-α production by PBMC from tuberculosis patients was associated with a comparatively weaker elevation of PGE2 synthesis which did not parallel fever or weight loss. In vitro treatment of control PBMC with the tuberculin purified protein derivative (PPD) promoted an increased TNF-α production which was similar to that of untreated PBMC from tuberculosis patients. Thus, the increased TNF-α production in tuberculosis could be explained by the in vivo exposure of PBMC to mycobacterial antigens. In contrast, the concentration of PGE; was weaker in the medium of untreated PBMC from tuberculosis patients than in the medium of PPD-treated control PBMC, suggesting that PGE, synthesis by PBMC was limited in tuberculosis by unidentified factors.
Natural killer cell activity, interferon-gamma and antibodies to herpes viruses in patients with Behçet's disease
Authors: HAMZAOUI K , AYED K , SLIM A , HAMZA M , TOURAINE J
Published: July 2010

Interferon-gamma (IFN-γ) titres in 20 patients with active Behçet's disease were examined and compared with those of 20 normal donors. Sera from Behçet's disease patients revealed an IFN-γ increase but no correlation between IFN level and natural killer (NK) activity. The analysis of lymphocyte subsets by monoclonal antibodies registered an increase of CD8+ T subpopulation and cells co-expressing CD8+ -Leu 7a+ markers. Moreover, a high number of cells expressing CD25+ and HLA-DR+ phenotype has been noted in patients with active Behçet's disease. Serological analysis showed a high level of IgG antibodies to HSV-1. The increase of IFN-γ litre, the high number of activated T cells and the increasing level of IgG antibodies to HSV-1 are important manifestations during the active stage of Behçet's disease. These findings are discussed in relation to the immunopathogenesis of Behçet's disease.
Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture
Authors: MCLACHLAN S M , TAVERNE J , ATHERTON M C , COOKE A , MIDDLETON S , PEGG C A S , CLARK F , SMITH B REES
Published: July 2010

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-α(TNF-α), interferon-gamma (IFN-γ), interleukin-1 (IL-1 α and β) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-α nor IL-2 affected thyroid cell survival, IFN-γ was usually inhibitory and IL-lα slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-γ, IL-2 and TNF-α inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-γ may inhibit thyroid cell proliferation and TNF-α, IFN-γ and IL-2 may down-regulate thyroid autoantibody synthesis.
Cloning and nucleotide sequence of cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from patients with systemic lupus erythematosus
Authors: NIKAIDO T , SHIMADA K , SHIBATA M , HATA M , SAKAMOTO M , TAKASAKI Y , SATO C , TAKAHASHI T , NISHIDA Y
Published: July 2010

Patients with systemic lupus erythematosus (SLE) produce autoantibodies against a variety of nuclear antigens including Ki antigen. Although anti-Ki autoantibodies were found in a significant number of SLE patients, the nature of Ki antigen is poorly characterized. By using anti-Ki serum as a probe we have cloned a bovine cDNA directing the synthesis in Escherichia coli of a polypeptide immunologically indistinguishable from the authentic Ki antigen. A homologous human cDNA was also cloned and its nucleotide sequence predicted the entire primary structure of a novel nuclear protein with a molecular weight of 29 508 and with highly hydrophilic and weakly acidic character. The gene is highly conserved not only in the coding region but also in the 3′-untranslated region. The bacterially produced Ki antigen would be valuable for diagnosis of SLE.
Lymphoid and non-lymphoid cells in the adenoid of children with otitis media with effusion: a comparative study
Authors: VAN NIEUWKERK E B J , DE WOLF C J M , KAMPERDIJK E W A , VAN DER BAAN S
Published: July 2010

We characterized on immuno- and enzymecytochemical level the lymphoid and non-lymphoid cells in the adenoid of children with upper respiratory tract infections (URI) and otitis media with effusion (OME) and compared these with the adenoid of children with URI without OME and with the adenoid of ‘healthy’ children and adults. Besides macrophages and dendritic cells we also showed the presence of MHC class II positive, ciliated, epithelial cells. These non-lymphoid cells were present in all adenoids. However, their number was less than 1% of all cells. We found no difference in lymphocyte subsets from children with URI+OME compared with those from children with URI alone. These two groups showed a significant decrease of CD8-positive (suppressor/cytotoxic) cells and a slight increase in CD22-positive B cells in comparison to ‘healthy’ children. No difference was found in percentages of CD4-positive (helper/inducer) cells. The localization of the lymphoid subsets in adenoids of children with URI and/or OME did not differ from those of ‘healthy’ children and adults.
C4a anaphylatoxin levels as an indicator of disease activity in systemic lupus erythematosus
Authors: WILD G , WATKINS J , WARD A MILFORD , HUGHES P , HUME A , ROWELL N R
Published: July 2010

The use of a synthetic protease inhibitor, nafamstat mesilate, has enabled reliable estimations of in vivo complement activation to be made in systemic lupus erythematosus (SLE). Elevation of C3a anaphylatoxins was found in two out of 24 patients and elevation of C4a anaphylatoxins was found in 20 out of 24 patients, confirming that complement activation, predominantly by the classical pathway, is a common occurrence in the disease. Significantly higher levels of C4a anaphylatoxin were found in 16 patients, with more aggressive disease requiring supplementary treatment with azathioprine, while the remaining eight patients, with less severe disease, required purely steroid therapy. Very strong associations between elevated C4a anaphylatoxins and raised DNA antibody titres, Clq binding activity and low complement C4 levels were also observed, suggesting that anaphylatoxin measurement may be a sensitive additional method for monitoring disease activity in SLE.
Selective concentration of IgD class-specific antibodies in human milk
Authors: LITWIN S D , ZEHR B D , INSEL R A
Published: July 2010

The participation of human IgD class antibody in local immune responses of breast tissue was studied by analysing the sera-to-milk ratios of total IgD, IgM, IgA, IgG isotypes and albumin found in matched samples, and by analysing the sera-to-milk (S/M) ratios of IgD, IgM, IgA, IgG antibodies against Haemophilus influenzae capsular polysaccharide (PRP), phosphorylcholine, tetanus and in some cases diphtheria antigens. The study group consisted of eight women immunized during pregnancy with PRP, and control, unimmunized women. Albumin, and total IgG showed high S/M ratios. IgA had a low S/M ratio as expected, consistent with reports that IgA is locally concentrated. Total IgD and IgM isotype ratio values were intermediate between IgG and IgA suggesting they were selectively concentrated in breast fluids due to local production or transport mechanisms, or both. Ratios for specific antibodies of IgA and IgM isotypes and for total IgA and IgM isotype showed parallel data. Among the IgD antibodies, those specific for PRP and phosphorylcholine suggested a higher degree of selective concentration as compared with tetanus antigen. In the group of unimmunized women, although selective concentration of total IgD was observed, specific antibody studies were inconclusive due to the low milk IgD antibody levels encountered. The results indicate that IgD (and also IgM) may participate in local immune responses of human breast tissues and fluids; possibly influenced by the nature of the antigen, the state of immunization and the hormonal environment (pregnancy).
Identification of protein components reactive with anti-PM/Scl autoantibodies
Authors: GELPI C , ALGUERÓ A , MARTINEZ M ANGELES , VIDAL S , JUAREZ C , RODRIGUEZ-SANCHEZ J L
Published: July 2010

The PM/Scl antigen from mammalian cells has been characterized as a nucleolar and nucleoplasmic molecular complex containing at least 16 polypeptides ranging in molecular weight from 110 to 20 kD. Of these polypeptides. we have found those of68, 39 and 20 kD to be in a phosphorilated form. Whereas the entire complex was precipitated by all the anti-PM/Scl sera tested, in immunoblots the antibodies specifically recognized determinants on the 110-kD protein. This protein was immunoprecipitated more preferentially from nuclcoli extracts than from total cell extracts. Moreover, this protein disappeared from the immunoprecipitates when treated with DNAse. Likewise, the immunoblol reaction of the specific antibodies with the 110-kD protein was abolished by treatment of the extracts with DNAse and trypsin, and was resistant when extracts were treated with RNAsc. Affinity-purified antibodies from this protein selectively stained the nucleoli and the nucleoplasm of the mammalian cells. Moreover, when the cultured cells used in immunofluorescence were treated with DNAse, the affinity purified antibodies from the 110-kD protein gave negative fluorescence. However, when whole anti-PM/Scl sera were used, a nucleolar and nucleoplasmic staining was found. We conclude that the 110-kD protein has at least one of the autoimmunogenic epilopes of the PM/Sel antigen, recognized by all anti-PM/Scl sera tested. Other epitopes differing in their DNAse sensitivity may also be present in the PM/Scl antigen.
Subclass distribution of IgG antibodies to the rat oesophagus stratum corneum (so-called anti-keratin antibodies) in rheumatoid arthritis
Authors: VINCENT C , SERRE G , BASILE J-P , LESTRA H C , GIRBAL E , SEBBAG M , SOLEILHAVOUP J-P
Published: July 2010

Serum IgG, labelling the stratum corneum of the rat oesophagus epithelium, so-called anti-keratin antibodies (AKA) constitute the most specific marker for the diagnosis of rheumatoid arthritis. In this study, we investigated 31 IgG AKA-positive rheumatoid sera and 21 control sera from patients with non-rheumatoid inflammatory rheumatic diseases. The serum level of IgG1,2,3 and 4 was determined by radial immunodiffusion and the subclass distribution of IgG AKA by a three-step semi-quantitative immunofluorescence assay using standard monoclonal antibodies specific for each of the four human IgG subclasses. In the rheumatoid sera, the serum level of IgGl was found to be significantly increased and the level of IgG2 significantly decreased with regard to the control sera, while the levels of IgG3 and 4 as well as total IgG were in the normal range. IgGl,2,3, and 4 AKA were detected in 27 (87%), 6 (19%), 4 (13%) and II (35%) of the 31 rheumatoid sera, respectively, and were found to be independent of the clinical and biological indices of the disease. In spite of inter-individual heterogeneity, two predominant profiles were distinguished: IgGI (alone) and lgG(I +4), which together represented 18 sera (58%). The large predominance of IgGI AKA and the quasiabsence of IgG2 AKA suggest that the recognized antigen may be partly comprised of protein. Moreover, the high frequency of occurrence of IgG4 AKA might result from chronic exposure to the eliciting antigen, which could be a genuine autoantigen since we demonstrated that it is also present in the stratum corneum of human epidermis.
Effects of neutral pepsin on the deposition of dietary antigens in glomeruli from IgA nephropathy
Authors: SATO M , KOJIMA H , KINO K , KOSHIKAWA S
Published: July 2010

Recently we reported on the participation of dietary antigens in the pathogenesis of IgA nephropathy. Particularly, the glomerular deposition of dietary antigens (casein, soybean protein, rice protein) was observed in some cases with IgA nephropathy. It is known that human urinary pepsin (HUP) prepared from the urine of a healthy adult acts as an immune complexase at neutral pH. In this study, the effects of incubation with HUP on the glomerular deposition of dietary antigens was investigated by an immunofluorescence technique in 48 patients with IgA nephropathy. four patients with Henoch Schonlein purpura nephritis and 69 with other glomerulonephritides. In 33 cases (68·8%) soybean protein and in 12 (25·0%) casein was found in biopsies of patients with IgA nephropathy. After a 3-day incubation with HUP there was no change in the deposition of IgA in 12 cases, a decrease in 22 and complete elimination in 14 cases. The deposits of soybean protein increased in 12 cases, did not change in 20, decreased or was abolished in five and was positively converted in 11. Exceptionally, in five patients treated with HUP, the deposition of soybean protein appeared concomitantly with the disappearance of IgA deposits. Such a concomitance did not occur either in other glomerulonephritides or in studies of other dietary antigens. Thus it appears that soybean protein may be deposited in the glomerular mesangium as an antigen in some patients with IgA nephropathy.
Frequencies of interleukin-5 mRNA-producing cells in healthy individuals and in immunoglobulin-deficient patients, measured byin situhybridization
Authors: SMITH C I E , MÖLLER G , SEVERINSON E , HAMMARSTRÖM L
Published: July 2010

Interleukin-5 (IL-5) has previously been demonstrated to enhance immunoglobulin synthesis, especially IgA. Thus, it could be hypothesized that a defect production of IL-5 may cause immunoglobulin deficiency. We have analysed the frequency of IL-5 mRNA-producing cells in healthy adults and in patients with common variable immunodeficiency or selective IgA deficiency. Unstimulated lymphocytes were rarely found to synthesize IL-5 as measured by in situ hybridization. However, pokeweed mitogen and several other activating ligands induced the synthesis of IL-5 mRNA in peripheral blood and spleen lymphocyte cultures. After pokeweed mitogen activation, the number of IL-5 mRNA-producing cells most often peaked on day 3 with a maximal frequency of around 1–2% of mononuclear cells. In a kinetic study we were unable to detect any peak frequency differences between healthy controls (mean 0.44%) and 20 patients (mean 0.58%), Thus, although IL-5 has been reported to be an important regulator of IgA synthesis, a defect production does not seem to be the underlying mechanism in human immunoglobulin deficiency.
Effects of interleukin-2 and interleukin-2-activated cells onin vitromyelopoiesis
Authors: CLERIGUE M , PISA P , TSAI L , HANSON M
Published: July 2010

Lymphokine-activated killer (LAK) cells from human peripheral blood mononuclear cells cultured with recombinant interleukin-2 (IL-2) have been used clinically in adoptive immunotherapy for cancer patients. To study the influence of LAK cells and IL-2 on haematopoiesis, an in vitro assay system for colony formation of granulocyte-macrophage progenitor cells (GM-CFC) was used. LAK cells from cultures of either human peripheral blood (PB) or human bone marrow (BM) mononuclear cells were both inhibitory to allogeneic BM-derived GM-CFC. Inhibitory activity could be transferred with supernatants from co-cultures of LAK cells and BM targets, but also from the IL-2 activated PB- or BM-derived cells alone. The inhibitory activity from the initially non-cytotoxic/non-inhibitory BM population was rapidly induced by IL-2 activation, and preceded the generation of cytotoxic LAK cells in the culture. These experiments show that inhibition of haematopoietic progenitor cells by IL-2 is not dependent on generation of cytotoxic LAK cells, but rather the result of IL-2-induced cytokine production. We conclude that the synergistic action of interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) may contribute to inhibition, but that also other cytokines are responsible for the observed inhibition of BM-derived GM-CFC.
Low-dose exposure to antigen induces sub-clinical sensitization
Authors: FRIEDMANN P S , REES J , WHITE S I , MATTHEWS J N S
Published: July 2010

We examined the effects of a small initial sensitizing dose of antigen (dinitrochlorobenzene, DNCB) on the subsequent response to a second, defined sensitizing stimulus. The second stimulus was actually the regimen of four doses of DNCB (3.125, 6.25, 12.5, and 25 μg; total 46.9 μg) normally used as the elicitation challenge. In two separate experiments 13 and 18 control subjects received an initial ‘challenge’ with the four doses to induce sensitivity, and 4 weeks later their responses were determined with a second, elicitation challenge. Two groups of 12 and 15 experimental subjects received an inital dose predicted to induce clinically detectable sensitivity in 50% or 25%, respectively. Four weeks later, their responsiveness was determined with quantitative challenge and the subjects who gave no response received a further challenge 4 weeks later. Their responses, compared with those from the control subjects, were augmented, indicating that sub-clinical priming of the immune system had indeed occurred.
Intra-epithelial lymphocytes: interferon-gamma production and suppressor/cytotoxic activities
Authors: EBERT E C
Published: July 2010

Human intraepithelial lymphocytes (IEL) proliferate minimally in response to phytohaemagglutinin (PHA), but produce as much interleukin-2 (IL-2) as do peripheral blood lymphocytes (PBL). The addition of sheep erythrocytes during activation of IEL with PHA markedly augments both T cell functions. This study evaluates the ability of IEL to produce interferon-gamma (IFN-γ) and to develop suppressor and cytotoxic activities when stimulated with mitogens in the presence or absence of sheep erythrocytes. PHA-activated IEL produced as much IFN-γ as did PHA-activated peripheral blood CD8+ T lymphocytes. IEL activated by concanavalin A (Con A) demonstrated less suppressor activity directed against T cell proliferation than did Con A-activated peripheral blood CD8+ T lymphocytes. IEL generated less mitogen-induced cellular cytotoxicity and lymphokine-activated killer cell activity than did peripheral blood CD8+ T lymphocytes. The addition of sheep erythrocyte lysates during mitogen stimulation of IEL markedly enhanced their proliferation and lymphokine production but did not affect their suppressor or cytotoxic activities.
Macrophage activation in falciparum malaria as measured by neopterin and interferon-gamma
Authors: BROWN A E , WEBSTER H K , TEJA-ISAVADHARM P , KEERATITHAKUL D
Published: July 2010

Macrophage activation during acute falciparum malaria in 71 Thai adults was investigated by measuring urinary neopterin and serum interferon-gamma (IFN-γ). Neopterin, a product of IFN-γ-activated macrophages, was elevated in 94% of patients upon admission (day 0, prior to treatment) and in all at some time during the period of study. Neopterin levels tended to rise further (days 1–5) before falling back towards the normal range as patients recovered following effective chemotherapy (days 6–8). IFN-γ was measured in 32 patients and found to be directly related to neopterin concentration. Both neopterin and IFN-γ values were highest in patients experiencing a first malaria infection. Among those with histories of prior malaria, neopterin and IFN-γ levels were inversely related to the number of previous infections. Morbidity, as assessed by degree and duration of fever, was directly related to neopterin concentration. This longitudinal study quantitatively describes the extent and duration of macrophage activation in falciparum malaria. The data also suggest that with repeated malaria infection and antigen exposure, there is a progressive decrease or possibly suppression of the T cell-macrophage interaction mediated by IFN-γ.
Induction and increase of HLA-DR antigen expression by immune interferon on ML-3 cell line enhances the anti-HLA-DR immunotoxin activity
Authors: CHIRON M , JAFFREZOU J P , CARAYON P , BORDIER C , ROUBINET F , XAVIER C , BRANDELY M , LAURENT G
Published: July 2010

In order to evaluate the impact of induction and increase target antigen expression on immunotoxin potency, we measured the potentiating effect of recombinant immune interferon-gamma (rIFN-γ) on the cytotoxicity of an anti HLA-DR ricin A-chain immunotoxin (2G5 RTA-IT) on the myeloid cell line ML-3. After 48 h of incubation with rIFN-γ (500 U/ml) the percentage of 2G5-positive cells increased from 40%. to 79%, and the 2G5 mean density was enhanced by 10-fold (11000 versus 110 000 molecules/cell). Concurrently, rIFN-γ pretreatment induced a dramatic improvement of 2G5 RTA-IT dose-effect cytotoxicity, as well as immunotoxin cytotoxicity kinetics. When 2G5 RTA-IT was used at the optimal dose of 10-8m (the maximum dose which avoided non-specific ricin A-chain cytotoxicity), the immunotoxin-induced cell kill increased with the percentage of DR-positive ML-3 cells according to a similar linear-logarithmic function of rIFN-γ concentration. Moreover, in the same range of rIFN-γ concentrations, the killing values and the percentage of DR-positive M L-3 cells were similar if not identical. These findings imply that the enhancement of 2G5 RTA-IT cytotoxicity by rIFN-γ is mainly related to the rIFN-y 2G5 antigen induction on HLA-DR negative cells when immunotoxin was used at 10-8m. Furthermore. 2G5 RTA-IT dose effect cytotoxicity on DR-expressing ML-3 cells, when used at lower concentrations, was also increased by rIFN-y in a dose-dependent manner. This result suggests that for immunotoxin concentrations close to the limiting membrane saturation dose (10-10 M). rIFN-y may not solely act by inducing HLA-DR expression on DR-negative ML-3 subpopulation but also by increasing individual cellular DR density on DR expressing ML-3 cells. Finally, our study showed that immunotoxin potency on malignant cell populations which display an heterogeneous antigen expression, could be greatly improved by the use of rIFN-γ.
Diverse target antigens recognized by circulating antibodies in anti-neutrophil cytoplasm antibody-associated renal vasculitides
Authors: SAVIGE J A , GALLICCHIO M , GEORGIOU T , DAVIES D J
Published: July 2010

Antibodies that arc directed against cytoplasmic constituents of neutrophils and monocytes (anti-neutrophil cytoplasm antibodies, ANCA) have been described in Wegener's granulomatosis, microscopic polyarteritis (MPA) and some cases of segmental necrotizing glomerulonephritis (SNGN). Other antibodies occasionally described in Wegener's granulomatosis and MPA include anti-nuclear antibodies (ANA) and anti-glomerular basement membrane (GBM) antibodies. We have studied the diversity of the corresponding antigens in ANCA-associated renal diseases. Sera from 46 patients with active histologically proven Wegener's granulomatosis, MPA and SNGN were tested for ANCA by indirect immunofluorescent examination of normal peripheral blood neutrophils. Thirty-four sera (74%) were positive; 16 were associated with diffuse cytoplasmic staining (cANCA) and 18 with perinuclear staining (pANCA). In addition, five demonstrated antineutrophil-specific nuclear staining (ANNA). On Western blotting of the neutrophil extract, five sera recognized a 29-kD molecule recently identified as neutrophil proteinase 3. Two sera with typical cANCA bound to molecules of 36, 38 and 116 kD and another to a molecule of 22 kD. The final serum associated with pANCA bound to a molecule of about 12 kD. Thirteen sera out of 46 (28%) tested in an ELISA contained anti-myeloperoxidase antibodies; 10 of these were associated with pANCA and two others with ANNA. Three sera of 17 (18%) tested contained anti-elastase antibodies; these also contained anti-myeloperoxidase antibodies and were associated with pANCA. However, eight sera with pANCA were negative for anti-myeloperoxidase antibodies and three of these were also negative for anti-elastase antibodies, suggesting further unidentified target antigen or antigens associated with the pANCA. Fifteen of the 34 sera positive for ANCA also demonstrated anti-nuclear staining on Hep-2 cells (53%) in a speckled, homogeneous, or nucleolar pattern. ANA were significantly associated with the presence of pANCA (P < 0.01), and levels of ANA and ANCA fell in parallel after treatment. One serum with a pANCA was also positive for anti-GBM antibodies. Inhibition studies using ELISAs for anti-GBM antibodies indicated that there was no cross-reactivity between target molecules recognized by these antibodies. The diversity of target molecules recognized by ANCA suggests that cross-reactivity with bacterial structures is less likely as the primary aetiological event in the development of these antibodies than tissue destruction; and that cross-reactivity with vascular endothelium is also unlikely as the pathogenetic basis of vessel disease.