bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumour Necrosis Factor‐α Induces Neutrophil‐Mediated Injury of Cultured Human Endothelial Cells
Authors: VON ASMUTH E. J. U. , LEEUWENBERG J. F. M. , VAN DER LINDEN C. J. , BUURMAN W. A.
Published: June 2006

We investigated the ability of TNF‐α to mediate damage of endothelial cells in the presence of neutrophils. by measuring detachment of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell detachment was increased from 5% to about 75% by the presence of I‐10 ng/ml TNF‐α during incubation with neutrophils, whereas negligible endothelial cell lysis was observed as measured by 51Cr release. TNF‐α was compared with the cytokines IL‐lα and IFN‐γ and with PMA and LPS. Both TNF‐α and PMA appeared to be strong triggers for neutrophil‐induced endothelial cell detachment, whilst reduced injury was seen after addition of IL‐lα and LPS. IFN‐γ did not induce endothelial cell detachment, but potentiated the effect of both TNF‐α and IL‐lα. TNF‐α‐induced endothelial cell detachment was neutrophil dependent, since pre‐incubation of neutrophils, but not pre‐incubation of endothelial cells with TNF‐α, caused endothelial cell detachment. Thus, TNF‐α‐induced increase in neutrophil‐adhesiveness of HUVEC was found not to be essential for endothelial damage. Pre‐incubation of neutrophils in suspension with TNF‐α induced rapid activation, followed by nearly complete deactivation of neutrophils, as measured by their capacity to induce detachment of endothelial cells after removal of TNF‐α.

These results indicate that local presence of TNF‐α might be critical in tissue or organ damage during early, neutrophil‐mediated inflammatory processes, independent of enhanced adhesiveness of endothelium for neutrophils.
Antigen Processing and Presentation by Small and Large B Cells
Authors: GONTIJO C. M. , MÖLLER G.
Published: June 2006

We have investigated the ability of different cells from non‐immunized mice of the BALB/c strain to present antigen to two ovalbumin‐specific I‐Ad‐restricted T hybridomas. Lipopolysaccharide‐activated B‐cell blasts were found to be the most efficient antigen‐presenting cells. Purified small and dense splenic B cells also stimulated the hybridomas although not to the same extent as the activated blasts, but comparable to non‐fractionated spleen cells. Glutaraldehyde‐treated B cells failed to present antigen, whereas F(ab′)2 anti‐mouse IgM‐treated B cells exhibited markedly increased ability lo present antigen. Using (low cytometry, we further purified the resting B cells by sorting the small lymphocytes to ensure that the ability of these cells to activate the hybridomas was not due to contamination with large non‐resting B cells. The sorted small B cells retained the ability of antigen presentation. Their resting state was confirmed by the fact that they did not incorporate [3H]‐thymidine as shown by autoradiographic analysis.
Human MHC Class I Antigens are Associated with a 90‐kDa Cell Surface Protein
Authors: FERM M. T. , GRÖNBERG A.
Published: June 2006

Human MHC class I proteins are expressed on almost all nucleated cells as a heavy chain (about 45 kDa) non‐covalently associated with β‐microglobulin (12 kDa), In this report we show that MHC class I (MHC‐I) proteins can also be associated with a 90‐kDa protein in the cell membrane.

Surface‐radiolabelled cells were treated with dithiobis succinimidyl propionate (DSP) in order to preserve multimer protein complexes during cell lysis. The lysates were immunoprecipitated and analysed by SDS‐PAGE and autoradiography, Immunoprecipitation of human MH C‐ I proteins co‐precipitated another protein of about 90 kDa in molecular weight p90. p90wasco‐precipitaled from all the MIIC‐I expressing cells tested: WIT. Raji. Molt‐4 and IFN‐γ treated K562, but not from untreated, MHC‐I negative K562. A 90‐kDa protein was also co‐precipitated with MHC‐I from fresh peripheral blood mononueiear cells(PBMC). Furthermore, p90 was co‐precipitated by different MoAbs lo the MHC‐1 heavy chain or β2‐microglobulin. bul not by control antibodies. Two additional co‐precipitaling proteins al 34 kDa and 28 kDa were seen in MHC‐I precipitates from Raji cells.

Our results suggest that MHC‐1 proteins and the 90‐kDa protein are associated in the cell membrane, probably by a close but weak, non‐covalent interaction. Two additional cell surface proteins at 34 kDa and 28 kDa seem to be MHC‐I associated on Raji Burkitt's lymphoma cells.
Lymphoproliferative Responses to Plasmodium falciparum Antigens in Children With and Without the Sickle Cell Trait
Authors: ABU‐ZEID Y A , ABDULHADI N. H. , HVIID L. , THEANDER T. G. , SAEED B. O. , JEPSEN S. , JENSEN J. B. , BAYOUMI R. A.
Published: June 2006

Blood mononuclear cells (BMNC) were isolated from sickle cell trait (HbAS) healthy donors and normal haemoglobin (HbAA) healthy donors resident in a P. falciparum endemic area of eastern Sudan. Blood samples were collected during the malaria season. BMNC were tested for their proliferative responses to phytohaemagglutinin (PHA), purified protein derivative of tuberculin (PPD) and to soluble P. falciparum antigens (SPAg). Higher responses to SPAg and PPD were observed in the HbAS children compared with the HbAA children, whereas no differences were observed among adults of the two phenotypes. Proliferative responses to PHA were comparable in all individuals tested.

The significance of these findings in relation to haemoglobin phenotype, age and the possible immunoregulatory mechanisms operating in HbAS and HbAA children during the malaria season is discussed.
Impaired Balance of Interleukin‐4 and Interferon‐y Production in Infections with Schistosoma mansoni and Intestinal Nematodes
Authors: ZWINGENBERGER K. , HOHMANN A. , DE BRITO M. CARDOSO , RITTER M.
Published: June 2006

In chronic infection with Schistosoma mansoni, IL‐4 and IFN‐γ are co‐modulated in opposite directions. This was shown when testing sera and cell culture supernatants from .11 Brazilian patients with schistosomiasis before, and three months after treatment with praziquantel. Thorough examinations were undertaken to account for polyparasitism with intestinal nematode infections involving tissue migrating larval stages that may induce analogous changes. Controls free of S. mansoni included a group (n = 17) matching the schistosomiasis patients’parasitation by intestinal nematodes and a group (n= 16) free of helminths other than T. trichiura. Serum lL‐4 was >30 pg/ml in 81% of schistosomiasis patients but in only 33 and 25%. respectively, of controls with and without intestinal nematodes. IL‐4 data correlated inversely with the mitogen‐induced if N‐y synthesis. Generation of IL‐4 in response to phorbol esters was related to the intensity of infection by schistosomes and intestinal nematodes. The parasitological status three months after therapy with cither praziquantel or mebendazole revealed a dichotomy: whereas the ratio of IFN‐7 lo IL‐4 generated in vitro was identical in uninfected controls and in patients who cleared the parasites, failure to eliminate the parasites was associated with lower IFN‐v/IL‐4 ratios generated in vitro.
A Novel Form of CD4 (L3T4) mRNA in the Murine Fetal Liver Results in Cell‐Surface Expression of the L3T4 Antigen
Authors: HAARS R. , CONRADT P. , MILTNER L. , WAGNER H.
Published: June 2006

The expression of the L3T4 antigen during ontogeny in the murine fetal liver has been investigated in parallel by northern blot analysis and cytofluorometry. The L3T4 gene is transcribed in the murine fetal liver in two polyadenylated mRNA species with the sin: of 3.5 kb and 3.7 kb. Whereas the 3.5‐kb mRNA is expressed from days 13 lo 18 of gestation, expression of the 3.7‐kb mRNA is found only from days 16 to 18 of gestation and thus appears to be developmentally regulated. Immunofluorescent staining of fractionated fetal liver cells from days 12 to 18 of gestation with the anti‐L3T4 antibody (GK i.5) provides evidence that cell‐surface expression of the L3T4 antigen on a subset of lymphohaematopoietic cells in the murine fetal liver is the product of a novel form of L3T4 mRNA with the size of 3.5 kb
Subclasses of IgG Anticardiolipin Antibodies in Patients with Systemic Lupus Erythematosus
Authors: TIIKKAINEN U. , WANGEL A. , APPLETON S. L. , ARTHUR D.
Published: June 2006

Antibodies directed to a co‐factor associated with negatively charged phospholipids, such as cardiolipin, occur in patients with systemic lupus erythematosus (SLE), and possibly more often in those with venous or arterial thrombosis, thrombocytopenia or recurrent fetal loss. They are also found in patients without any of these manifestations and their biological effect, if any, might thus be related to their IgG subclass. To investigate this possibility, we determined anticardiolipin antibodies (ACA) by enzyme immunoassay (EIA) using monoclonal antibodies (MoAb) against human IgG subclasses. A net absorbance of x +3 SD of the value of 30 blood donors was taken as the cut‐of point. The specificity of the assay was verified through inhibition experiments using cardiolipin micelles. Thirty‐three patients with SLE were studied, all of whom had been shown to have ACA by a point dilution screening assay. IgGI ACA were found in 85% of the patients, and ACA of the IgG2, IgG3 and IgG4 subclasses in 42%, 39%. and 15%. There was a significant correlation between the presence of IgG3 ACA and of anti‐DNA antibodies but none between subclass distribution and major clinical manifestations of SLE.
The Role of CD45RA on Human B‐Cell Function: Anti‐CD45RA Antibody (Anti‐2H4) Inhibits the Activation of Resting B Cells and Antibody Production of Activated B Cells Independently in Humans
Authors: MORIKAWA K. , OSEKO F. , MORIKAWA S.
Published: June 2006

Anti‐CD45RA antibody defined by anti‐2H4 monoclonal antibody has been reported to split CD4+T cells into two distinct subpopulations. CD45RA antigen is present on the surface of virtually more than 95% B lymphocytes in the purified tonsillar B‐cell preparations. We examined the role of CD45RA antigen on human B‐cell function using this antibody. The addition of anti‐2H4 to tonsillar B cells inhibited the proliferative response induced by Stapylocoocus aureus Cowan strain I(SAC) in a dose‐dependent manner, Kinetic analysis indicated that anti‐2H4 exerted its inhibitory effect when added within the first 24 h of culture initiation during a 72‐h culture period. Anti‐2H4 inhibited the transferrin receptor expression without interfering with the expression of the IL‐2 receptor on SAC‐stimulated B cells in a short‐term culture. Anti‐2H4 blocked the progress of SAC‐stimulated B cells from the G1 to S phase of the cell cycle. These events suggested that anti‐CD45RA MoAb inhibited the proliferative response by directly acting on B cells in the G1 phase. In addition anti‐CD45RA antibody also had a suppressive effect on early phase of B‐cell differentiation. This effect appeared to be independent of its suppressive effect on proliferation, because anti‐CD45RA did not inhibit the proliferative response of pre‐activated B‐cells with lymphokines. These studies suggested that the restricted epitope recognized by anti‐2H4 antibody may be directly involved in regulatory function on B cells.
T‐Cell Regulation of CD5+ B‐Cell Activity in Normal Mice
Authors: HUCHET R. , GRANDJON D. , HALLE‐PANNENKO O.
Published: June 2006

The differentiation of plaque‐forming cell (PFC) precursors against bromelain‐treated syngeneic erythrocytes (Br MRBC) into PFC induced in vitro by LPS is down‐regulated by nylon non‐adherent (nylon‐passed–NP) T cells and by nylon adherent (NA) T cells, NA T cells are more potent inhibitors than NP T cells. This regulatory activity of NA and NP T cells results from an interaction between CD4+ radioresistant and CD4+ radiosensitive T cells. Furthermore CD4+ T cells from the NA fraction but not from the NP fraction are activated cells: their inhibitory activity is abrogated after preincubation with cycloheximide. These results are discussed within the overall framework of T‐cell regulation of autoimmune anti‐Br MRBC B‐cell subsets.
Expression of the LFA‐1 β2 Integrin (CD11a/CD18) and ICAM‐1 (CD54) in Normal and Coeliac Small Bowel Mucosa
Authors: SMART C. J. , CALABRESE A. , OAKES D. J. , HOWDLE P. D , TREJDOSIEWICZ L. K.
Published: June 2006

The leucocyte adhesion molecules (β2 integrins) comprise CD11 α‐chains and a common β‐chain (CD18), CD11a (leucocyte Junction‐associated antigen 1, LFA‐1) is expressed by most T cells, and is involved in antigen presentation by macrophages via its counter‐receptor, intercellular adhesion molecule (ICAM‐1, CD54), By criteria of double‐label immunofluorescence of cryostat tissue sections, virtually all lamina propria T cells of the normal small bowel were found to express LFA‐1 strongly. By contrast, only 30‐60% of intra‐epithelial lymphocytes (IEL) expressed detectable LFA‐1 most of which were LFA‐Iweak and CD18‐ ICAM‐1 was expressed strongly only by vascular endothelium, in coeliac disease, there was a modest increase of diffuse ICAM‐1 expression in the lamina propria, mainly in the subepithelial zone, where ICAM‐1 + macrophages were occasionally seen. There was also a slight overall increase in CD11a expression by IEL, seen predominantly in surface epithelium and mainly by the CD4+ minority subset, but not by CD4‐ CD8‐ (TcR γδ+) cells. These data suggest that the LFA‐1/ICAM‐1‐dependent antigen presentation pathway is of minor importance to IEL in the normal small bowel, and does not assume a major role in coeliac disease.
A Murine Monoclonal Anti‐Idiotypic Antibody Detects a Common Idiotope on Human, Mouse and Rabbit Antibodies to Allergen Lol p IV
Authors: ZHOU E.‐M. , DZUBA‐FISCHER J. M. M. , RECTOR E. S. , SEHON A. H. , KISIL F. T.
Published: June 2006

A syngeneic mouse monoclonal anti‐idiotypic antibody (anti‐Id). designated as B1/I. was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV, This anti‐Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91, Observations that (i) the anti‐Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id anti‐Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combininfisite. These properties served to characterise HI 1 as an internal image anti‐Id, Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id anti‐Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti‐Id, Interestingly, the internal image anti‐Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV. different from that recognized by MoAb 91.
Localization of Circulating Immune Complexes from Patients with Rheumatoid Arthritis in Murine Spleen Germinal Centres
Authors: MAGEED R. A. , KIRWAN J. R. , HOLBOROW E. J.
Published: June 2006

In previous studies we have demonstrated high levels of rheumatoid factor (RF) and large‐size (> 22S) circulating immune complexes (CIC) in the serum of rheumatoid arthritis (RA) patients with extra‐articular disease. These findings were paralleled by a concurrent increase in the level of RF‐associated cross‐reactive idiotypes ICRI) and an apparent diversification of the RF repertoire detected in the serum of the same patients.

In the present study we examine the ability of CICs to activate the complement system in vivo, and Us possible influence on expanding the RF repertoire in RA patients with extra‐articular disease. Activation of complement by CICs is the key for germinal centre localization and long‐term retention of such complexes on the surface of follicular dendritic cells (FDC). and so provides a source for the selection of cells with high affinity receptors for IgG and leads to the establishment or immunological memory.

CICs containing different immunoglobulin isotypes and from different patients localized in mouse spleen germinal centres. However, intense localization was mainly seen for IgG‐containing complexes from the serum of patients with large‐size (> 22S) IgG RT complexes. T he ability of these complexes to localize in mouse spleen germinal centres was related to activation of the complement system via the classical pathway in the patients' sera. Localization of IgG complexes was significantly (P < 0.05) higher in sera from RA patients with extra‐articular disease than those with articular disease alone.

This study demonstrates the ability of large‐size (>22S) IgG IgM RF complexes to activate complement and suggests a possible role for such complexes in modulating the immune response to IgG in RA patients with extra‐articular disease.
Fine Molecular Specificity of Linear and Assembled Antibody Binding Sites in HIV‐1 p24
Authors: HAAHEIM L. R. , MASKELL J. P , MASCAGNI P. , COATES A. R. M.
Published: June 2006

A set of seven murine monoclonal antibodies were generated against a chemically synthesized 11‐kDa 104‐mer peptide covering the C‐terminal residues 270‐373 of the p24g.it; protein (HIV‐IBRU strain). All monoclonal antibodies recognized HIV‐LMN infected MOLT? cells by fluorescence und gave positive Western blot signals with viral gag peptides [p55 and/or p24), Oligopeptide binding regions were located with competitive enzyme‐linked immunosorbent assays. Detailed epitope scanning analyses (the Geysen technique) were performed by serological testing of the monoclonal antibodies against 99 overlapping hexapeptides which corresponded to the entire 104‐mer region. The antibodies bound to p24 peptide sequences located within the 275 2y3 and 351 36S regions. One antibody (LH 104‐B) which reacted with residues 357‐362 bound lo p55 alone. In contrast another antibody (LH 104–1). which recognized the residues 358–363. i.e. with five out of six residues in common with antibody LIIKM‐B for its epitope region, reacted exclusively with p24.

At least two of the antibodies (LH104‐C and ‐A) which bound to p24 alone, apparently recognized conformational epitopes, They gave positive reactions with the regions 288—293/351–356 and 284–289/351–356, respectively.

This work shows that chemical synthesis of large peptides is a viable alternative approach to immunochemical studies of viral proteins.
Expression of the CD2 Activation Epitope T11‐3 (CD2R) on T Cells in Rheumatoid Arthritis, Juvenile Rheumatoid Arthritis, Systemic Lupus Erythematosus, Ankylosing Spondylitis, and Lyme Disease: Phenotypic and Functional Analysis
Authors: POTOCNIK A. J. , MENNINGER H. , YANG S. Y. , PIRNER K. , KRAUSE A. , BURMESTER G. R. , BRÖKER B. M. , HEPT P. , WESELOH G. , MICHELS H. , TRUCKENBRODT H. , EMMRICH F. , KROCZEK R. A.
Published: June 2006

CD2R is an activation‐associated epitope unmasked by a conformational change of the CD2 cell‐surface glycoprotein. In spite of elaborate studies on the role of CD2 and CD2R in adhesion and stimulation of T cells in vitro, no instances of CD2R expression in vivo were known to date. We report high levels or CD2R observed on blood and synovial fluid T cells in rheumatoid arthritis and on peripheral blood T cells in juvenile rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and Lyme disease. In vivo, expression of CD2R was restricted to T cells, not limited to a particular T‐cell subset and not correlated with the expression of p55 interleukin 2R (IL‐2R) (CD25) or major histocompatibility complex (MHC) class II molecules. When stimulated to prolieration via CD2 or CD3, ex vivo CD2R+ T cells showed the same basic activation requirements as CD2R‐ T cells.
MPB 64 Possesses Tuberculosis‐Complex'‐Specific B‐ and T‐Cell Epitopes
Authors: ANDERSEN A. B. , LJUNGQVIST L. , HASLØV K. , BENTZON M. W.
Published: June 2006

We have developed monoclonal antibodies (MoAb) reactive with a protein from Mycobacterium tuberculosis of apparent molecular mass 24 kDa, This protein was shown lo he identical with MPB M (Harboe et al., [1]), The MoAb bound to four different epitopes of which two were restricted to the tuberculosis complex” and two were also found in mycobacteria not belonging to the “tuberculosis complex”, The cross‐reactive MoAb demonstrate that MPB M is present in more mycobacterial species than previously assumed. MPB 64 was show n lo induce strong delayed type hypersensitivity (Dth) reactions in out bred guinea pigs immunized with M tuberculosis and M. hovis bacille Calmette‐Guérin (BCG), No reaction was observed m animals immunized with mycobacteria not belonging to the “tuberculosis complex”, The Dth‐inducing capacity of MPB 64 was compared with that of another 24 kDa protein purified from W. merciless and of the previously described 38 kDa protein. The Dth responses lo these three antigens were further analysed in four inbred guinea pig strains. A genetic restriction of the ability of the animals to respond to MPB 64 as well as to the 38 kDa protein was observed.
Structural Studies of Immunoglobulins
Authors: PORTER RODNEY R.
Published: June 2006

In 1946, when I was starting work as research student under the supervision of Dr F. Sanger, the second edition of Karl Lansteiner's book” The Specificity of Serological Reactions’[I] reached England. In it was summarized the considerable body of information available on the range of antibody specificity and much of it was Landsteiner's own work or by others using his basic technique of preparing antibodies against haptenes and testing their ability to inhibit the precipitation of the antisera and t he conjugated protein. Also described in this book was the work in Uppsala of Tiselius and Pederson in collaboration with Heidelberger and Kabat in which they showed that all rabbit antibodies were in the y globulin fraction of serum proteins and that they had a molecular weight of 150.000. This combination of an apparently infinite range of antibody‐combining specificity associated with what appeared to be a nearly homogeneous group of proteins astonished me and indeed still does.
Phorbol Myristate Acetate Corrects Impaired NK Function of Old Mice
Authors: GHONEUM M. , SUZUKI K. , GOLLAPUDI S.
Published: June 2006

Previous studies indicated that natural killer (NK) activity in mice declined with age. In this report, we investigated the basis for the decreased activity of NK cells in older mice. Our results demonstrated that phorbol myristate acetate(PMA)–an activator of protein kinase C(PKC)– corrects the impaired NK function of older animals. The per cent increase of NK activity post treatment with PMA is 226 261 % at effector: target (E:T) ratio= 100:1 compared with control untreated cells. The effect of PMA was shown lo be dose dependent. A maximum increase in NK activity was achieved at approximately 10–15 nm PMA. Treatment with PMA does not result in increased binding of NK cells to YAC‐1 targets as demonstrated by single‐cell assay, in addition, treatment of young NK cells with l‐(5–isoquinolinesulphonyl)‐2–methylpiperazine dihydrochloride (H‐7). a very potent PKC inhibitor, significantly suppressed NK activity (65% of control). Therefore, we conclude that the age‐related decline in murine NK activity may reside in the early signalling events leading to triggering of PKC.
Dietary Supplementation with co‐3‐Polyunsaturated Fatty Acids Decreases Mononuclear Cell Proliferation and Interleukin‐1β Content but not Monokine Secretion in Healthy and Insulin‐Dependent Diabetic Individuals
Authors: MØLVIG J. , POCIOT F. , WORSAAE H. , WOGENSEN L. D. , BAEK L. , CHRISTENSEN P. , MANDRUP‐POULSEN T. , ANDERSEN K. , MADSEN P. , DYERBERG J. , NERUP J.
Published: June 2006

The effects of dietary supplementation with ω‐3‐polyunsaturated fatty acids (ω‐3‐PUFA) on the proliferative response of PBMC and on the secretion of monokines and arachidonic acid metabolites from PBMC and monocytes (Mo) from healthy subjects and patients with recent‐onset insulin‐dependent diabetes mellitus (IDDM) were examined. Three groups of eight to nine healthy individuals were randomized to either 2.0 g/day or 4.0 g/day of ω‐3‐PUFA devoid of vitamins A and D, or an isocaloric amount of placebo. Furthermore, eight patients with recent‐onset IDDM received 4.0 g/day of ω‐3‐PUFA. IL‐lβ production and TNF‐α secretion was determined before and after 7 weeks of treatment, and 10 weeks after withdrawal of treatment. Significant increases in platelet and PBMC membrane eicosapentaenoic acid was found in ω‐3‐PUFA‐treated individuals. ω‐3‐PUFA treatment significantly reduced the content of IL‐Ib in lysates of PBMC, but did not affect PBMC or Mo secretion of IL‐1β, TNF‐α or prostaglandin E2 (PGE2) or PBMC leukotriene B4 (LTB4) secretion in healthy subjects or in IDDM patients. A significant inhibition of the PHA‐stimulated. but not the spontaneous or PPD‐stimulated, proliferative response of PBMC was observed in healthy and diabetic subjects treated with (o‐3‐PUFA. No correlation was found between PHA‐stimulated PBMC proliferation and PBMC secretion of TNF‐α and IL‐1β. There were no significant differences in the spontaneous or the PPD‐or PHA‐stimulated proliferative responses of PBMC between diabetic and healthy individuals at entry. We conclude that although dietary supplementation with 4.0 g/day of ω‐3‐PUFA inhibits the proliferation of PBMC and reduces IL‐I immunoreactivity in PBMC and Mo, it does not alter monokine, PGE2 or LTB4, secretion in healthy or IDDM subjects.
Immunological Screening of a Glycoprotein Antigen Expressed by Zajdela Ascites Hepatoma Cells on Normal Rat Tissues and Tumour Cells
Authors: NATO F. , GOULUT C. , MIRSHAHI M. , BOURRILLON R.
Published: June 2006

Expression of the glycoprotein MII2 antigen originally identified in Zajdela ascites hepatoma cells was investigated in several normal rat tissues and in more or less differentiated tumours using biochemical and immunological approaches. SDS polyacrylamide gel electrophoresis followed by fluorography or immunoblotting with an antiserum raised against the purified MII2 antigen revealed that this antigen was absent from normal liver cells. ELISA assays, indirect immunofluorescence and immunoprecipitation experiments using the same antiserum showed that this glycoprotein was not expressed in various normal tissues such as liver, spleen, lung, pancreas, intestine and stomach, but it was unexpectedly detected in kidney and thymic (issues. However, the molecular weight of the antigens immunoprecipitated from kidney and thymus was lower than the one of MH2 (Mr of 60.000 versus 110,000 160,000 for purified MH2). No staining was observed in embryonic rat liver at 10 and 20 days of development.

Moreover, this antigen was present on the surface of Morris hepatoma 7777, another rapidly proliferating and poorly differentiated hepatocellular carcinoma. In contrast, this antigen was not detected on the surface of in vitro Zajdela hepatoma cells (ZHC) or of partially differentiated hepatomas (Faza) which have recovered some hepatic functions. In addition, the MH2: antigen was found on the human non‐hepatic HT‐29 tumour cell line, under its undifferentiated form (HT‐29 G’ subline). The possible relationships between the expression of this antigen and both the malignant transformation process and the differentiation process are discussed.
T‐Helper‐Cell Determinants in Protein Antigens are Preferentially Located in Cysteine‐Rich Antigen Segments Resistant to Proteolytic Cleavage by Cathepsin B, L, and D
Authors: MOURITSEN S. , MELDAL M. , RUUD‐HANSEN J. , WERDELIN O.
Published: June 2006

We report on a computer algorithm capable of predicting the location of T‐helper‐cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T‐cell determinants than susceptible segments.

From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1′, and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine‐containing T‐cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T‐cell determinants in the Ag with a relatively low number of false (positive) predictions.

Furthermore, we present a new principle for searching Ag for potential amphipatic α‐helical protein segments. Such segments accord well with empirically known T‐cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described.
There is No Correlation Between Function and Lymphokine Production of HBs‐Antigen‐Specific Human CD4+‐Cloned T Cells
Authors: TSUTSUI H. , MIZOGUCHI Y. , MORISAWA S.
Published: June 2006

The question whether antigen‐specific human CD4+ T cells can be classified on the basis of appropriate and fixed lymphokine production patterns and their corresponding functions still remains lo be elucidated. We generated ten CD4+ T‐cell clones specific for HBsAg from HBsAg‐positive but HBsAb‐negative individuals. Seven of these clones exhibited helper activity for HBsAb response, while the three other clones did not. Both helper‐ and non‐helper‐type T‐cell clones produced interleukin 4 (IL‐4) after antigenic stimulation. By stimulation with phytohaem‐agglutinin (PHA) plus phorbol myristate acetate (PMA). three of the seven helper‐type clones produced interleukin 2 (lL‐2) in addition toiL‐4. However, the other four helper‐type clones did not produce IL‐2 by such stimulation, although they continued the production of IL‐4. All non‐help)er‐type T‐cell clones produced a large amount of IL‐2. and some of them completely became an IL‐2 producer after certain stimulation. These results suggested that both helper‐ and non‐helper‐type CD4+ T‐cell clones specific for HBsAg might have no strict pattern of lymphokine production as m the TH1 /TH2 dichotomy of murine CD4+ T cells. The data also revealed that lymphokine‐producing capacity of individual cloned T cells is changeable depending upon the sort of activation.
Immunoglobulin‐Mediated Prevention of Autoimmune Diabetes in the Non‐Obese Diabetic (NOD) Mouse
Authors: FORSGREN S. , ANDERSSON Å. , HILLÖRN V. , SODERSTROM A. , HOLMBERG D.
Published: June 2006

We investigated whether the development of spontaneous T‐cell‐mediated type I diabetes in NOD mice is influenced by B cells and immunoglobulin (Ig). During the first 4 weeks of life. B‐cell development was suppressed by repeated administration of rabbit anti‐mouse IgM (RaIgM), while controls received polyclonal rabbit Ig (NRIg).A reduction in the incidence of diabetes, as well as in development of insulitis, was observed after either of these treatments. However, the effect on insulitis was more pronounced in mice treated with RaIgM compared with those treated with NRIg. Furthermore, while the optimal effect of NRig was obtained after a single injection at birth, the additional effect of RaIgM on development of insulitis was observed only after continued treatment for the first 4 weeks of life. Taken together these data suggest a possible role of Ig/B cells in the development of autoimmunity In the NOD mouse. The additional effect observed after continued suppression of the neonatal B‐cell development suggests that this population may contribute significantly to the establishment of an auto‐aggressive lymphocyte repertoire in the NOD mouse.
Hidden Rheumatoid Factor and Wa Idiotype Expression in Juvenile Rheumatoid Arthritis
Authors: ILOWITE N.T. , WEDGWOOD J.F. , MOORE T.L. , RAMAKRISHNAN T. , BONAGURA V. R.
Published: June 2006

The major rheumatoid factor cross‐reactive ideotype, which was defined by prototypic monoclonal igM rheumatoid factors from Waldenstrom's macroglobulinaemia patient Wa, is present on 60% of all monoclonal IgM RF paraproteins. One‐third of patients with Juvenile rheumatoid arthritis (JRA), who are seronegative for classic IgM rheumatoid factor (RF). express the Wa idiotype in high titre in their sera. To determine if the Wa idiotype is present on hidden rheumatoid factors in JRA patient sera, we studied hidden RF expression by both ELISA and haemolytic assay techniques. The majority of JRA sera with increased concentrations of the Wa idiotype did not have increased RF activity nor hidden RF activity. In some JRA patients, the Wa Idiotype may he expressed on a parallel set of immunoglobulin molecules, related to RFs. hut lacking the ability to bind to IgG.
Functional Epitope Analysis of the Human CD 11 a/CD 18 Molecule (LFA‐1, Lymphocyte Function‐Associated Antigen 1) Involved in HIV‐1‐Induced Syncytium Formation
Authors: VERMOT‐DESROCHES C. , RIGAL D. , ESCAICH S. , BERNAUD J. , PICHOUD C. , LAMELIN J.‐P. , TREPO C.
Published: June 2006

After binding lo the CD4 receptor, the human Immunodeficiency virus I HIV‐I)may enter the T cell and induce the formation of multinucleated giant cells (syncytia). As well as the CD4 molecule, other molecules, such as the lymphocyte function‐associated antigen 1(LFA‐1. CD11a/CD 18) have been shown to be involved in HIV‐l‐mediated cell fusion.

This study was designed to define regions on the human CD1la/CD18 molecule important for the HIV‐l‐induced syncytium formation A CD11a/CD18 MoAb panel discriminating at least five distinct and spatially distant domains of the LFA‐l molecule was used. Comparison of the functional activity of different MoAbs demonstrated that all epitopes of the LFA‐1 molecule were not of equal importance m HIV‐1‐induced syncytium formation between H9.III cells chronically infected with HIV‐1 and uninlecledCD4+ Sup T1 cells. We also demonstrated that CD11a/CD18 MoAbs inhibit syncytia formation only at the level of the uninfected Sup T1 cells, suggesting that the LPA‐1 molecule expressed on Sup T1 ceils interacts with ligand(s) expressed on the infected H9. III cells. Two potential LFA‐1 receptors on the H9.III cells were tested: t he ICAM‐1 molecule (intercellular adhesion molecule 1.CD54)and the HIV‐1 transmembrance glycoprotein41 (gp4l), A CD54 MoAb (84H10) partially inhibited syncytia formation, thus demonstrating the involvement of the ICAM‐1 molecule in the HIV‐l‐mediated cell fusion. However, the CD11a/CD18 MoAbs do not inhibit binding of the viral envelope glycoprotein gp41 to the cell surface, irrespective of the MoAb concentration used.

Although we have not been successful in identifying all candidate fusion receptors for the LFA‐1 molecule, these data suggest that some LFA.‐1 regions are important for syncytium formation and. therefore, in the cell‐to‐cell transmission of virus and in the spread of infection.
Cell Cycle‐Dependent Expression of CD4 Antigen in a Monocytoid Cell Line
Authors: MARTIN C. F. C. , CORREALE J. , KAM‐HANSEN S. , EHRNST A.
Published: June 2006

The CD4 molecule has several biological functions, physiologically as a receptor for major histocompatibility complex class II molecules on antigen‐presenting cells, and pathologically as a receptor for human immunodeficiency virus (HIV) by its binding to the HIV envelope glycoprotein gp 120 The frequency of’CD4+ cells has been shown to correlate positively with both susceptibility and cytopathogenic effect by HIV. To determine if CD4 expression varied during the cell cycle, a CD4‐cxpressing monocytoid cell line. U 937 clone 16, was synchronized with regard lo cell growth. The CD4 antigen was analysed with regard to expression, density and rate of reappearance after treatment with trypsin during the different phases of the cell cycle. The CD4 reappearance rate was found to be maximal during the S phase. This was followed by an increased expression and density in the late S G2 phase. Thus a cell cycle‐dependent expression of CD4 molecules on the cell surface was observed.
Non‐Immune Binding of Human Protein Fv to Immunoglobulins from Various Mammalian and Non‐Mammalian Species
Authors: BOUVET J.‐P. , PIRES R. , CHARLEMAGNE J. , PILLOT J. , ISCAKI S.
Published: June 2006

Reactivity of the secretory protein Fv with immunoglobulins (Ig) from various species of vertebrates was investigated. Binding was observed throughout all taxonomic classes: mammalian, avian, reptilian, amphibian and fish. Contrasting with tins wide spectrum, no significant binding was found with most mammalian ungulates, such as horse (Perissodactl), cow, sheep and goat (Artiodactyls), Nevertheless disruption of’the hydrogen bonds of Ig allowed these non‐reactive molecules lo bind. Such a conserved reactivity dining evolution, and our previous data on the effect of the cleavage of the intra‐chain disulphide bonds, suggest that protein Fv recognizes a discontinuous framework structure involving both the FRI and FR3 regions in the variable domain of the heavy chain of Ig.
Human Monoclonal Antibodies against Blood Group Antigens Preferentially Express a VH4‐21 Variable Region Gene‐Associated Epitope
Authors: THOMPSON K. M. , SUTHERLAND J. , BARDEN G. , MELAMED M. D. , RANDEN I. , NATVIG J. B. , PASCUAL V. , CAPRAJ J. D. , STEVENSON F. K.
Published: June 2006

An anti‐idiotypic antibody has been raised which recognizes human immunoglobulins with cold agglutinin activity of anti‐I/i specificity. The pattern of reactivity of the antibody indicates that the structural basis for the epitope is located in the VH4‐21 gene segment of the VHIV family, which is preferentially utilized by these cold reactive antibodies.

Using this antibody, epitope expression was investigated in a panel of 72 human monoclonal allo‐antibodies specific for human blood group antigens, as compared with a control panel of 39 randomly selected human monoclonal IgM antibodies of unknown specificities. The anti‐blood group panel included 44 IgM and 28 IgG monoclonal antibodies against a variety of blood group antigens including the A antigen, Rh C, c, D, E, e, G antigens, and the Kidd antigens Jka and Jkb. The epitope was expressed by 64% (28/44) of the IgM anti‐blood group antibodies and by 21 % (6/28) of the IgG antibodies, but by only 7.7% (3/39) of the control IgM antibodies.

These data indicate that the human alloimmune response to blood group antigens is biased in the use of VH gene families, with a preference for the VH4‐21 gene segment of the VHIV family, or closely related gene segments. The fact that this mirrors the findings for the autoimmune cold agglutinins suggests a link in immunoglobulin gene usage between antibodies against structurally diverse antigens on the red cell surface.
Quantitative and Qualitative Analysis of Human IgG Subclass Specific mRNA Using Solution Hybridization
Authors: SIDERAS P. , NILSSON L. , ISLAM K. B. , ERICSSON H. , HAMMARSTRöM L. , SMITH C. I. E.
Published: June 2006

Four IgG subclasses have been identified in the human system. Despite the tact that they exhibit differences in their functional properties, antigenic properties and chemical composition and demonstrate age‐related shifts in their expression, the genes coding for their constant regions show extensive homology at the nucleotide level (> 95%). The only parts of the Cγ genes that show significant variation among the different IgG subclasses are the exons coding for their hinge regions (< 60%). Taking advantage of such sequence variation, we have developed specific RNA probes which allowed us to analyse the expression of the Cγ1. Cγ2, Cγ3 and Cγ4 genes al the mRNA level using solution hybridization. We have defined optimal conditions that allow the detection of picogram levels of IgG subclass specific mRNA, while keeping the cross‐reactivity between the different probes below 2%. This approach will significantly facilitate studies aiming at characterizing the molecular mechanisms regulating the expression of the four human IgG heavy chain constant region genes.
Flow Cytometric Analysis of Natural Interferon‐α Producing Cells
Authors: SANDBERG K. , ELORANTA M‐L. , JOHANNISSON A. , ALM G. V.
Published: June 2006

Herpes simplex virus‐infected cells induce high interferon‐a (IFN‐α) production in infrequent cells among peripheral blood mononuclear cells (PBMC), designated natural IFN‐a producing (NIP) cells. The properties of such NIP cells were compared with defined populations of leucocytes by means of flow cytometric analysis and sorting. The NIP cells are characterized as a discrete population of cells with high forward and low to intermediate orthogonal light scattering, similar to that of early progenitors of myeloid and lymphoid cells. However, they appear lo lack the stem cell‐associated molecule CD34. Furthermore, NIP cells cannot be localized to the myeloid line of cell differentiation, because they do not express the CD33. CD 13, CD11b, CD15 or CD14 antigens. Neither do they express CDIO and CD19 antigens which are present in all stages of B‐cell differentiation plasma cells excepted, nor CD7 antigens expressed on early T cells. In combination with previous results, our data support the view that the NIP cell is a unique and distinct cell type in peripheral blood, possibly with a physiological role in the defence against certain viral infections.
Formation of Covalent C3b‐Tetanus Toxin Complexes: a Tool for the In Vitro Study of Antigen Presentation
Authors: VILLIERS* M.B. , VILLIERS C. L. , WRIGHT J. F. , MAISON C. M. , COLOMB M. G.
Published: June 2006

A novel method is described for the formation and purification of covalent complexes between the complement component C3b and an antigen (tetanus toxin. TT). using purified proteins in fluid phase. C3b is generated in situ by tryptic cleavage or C3 after co‐precipitation of C3 and TT in the presence of polyethylene glycol. Various parameters were analysed to optimize complex formation: under conditions which minimized the formation ofcovalentC3bmultimers, 30% and 8% respectively ofC3band TT were incorporated into covalent one‐to‐one complexes which were purified using gel filtration chromatography. The linkage WAS localized between the α chain of C3b and either the H or L chain of TT; n required the in situ formation of C3b and was partially destroyed by 1 M hydroxylamine. Spontaneous dissociation of the complex could be partly avoided by HgCl2, a thiol reagent which inhibits the esterase‐like activity of bound C3b, These findings suggest the involvement of the reactive carbonyl of nascent C3b with hydroxyl groups of TT, Such C3b TT complexes provide a defined tool to analyse the influence of antigen‐bound C3bon antigen addressing and intracellular processing by antigen‐presenting cells.
Macromolecular Properties of Glycosaminoglycans in Primary AL Amyloid Fibril Extracts of Lymphoid Tissue Origin
Authors: STENSTAD T. , MAGNUS J. H. , KOLSET S. O. , CORNWELL G. G. , HUSBY G.
Published: June 2006

We have previously demonstrated the presence of glycosaminoglycans (GAGs) in water extracts of secondary A A amyloid fibrils. In the present study we isolated significant quantities of GAGs from fibril extracts of immunoglobulin light chain (AL) type derived from the spleens from two patients afflicted with primary amyloidosis. Employing ion‐exchange chromatography and gel filtration subsequent lo various specific chemical and enzymalic treatments, different types of high molecular weight GAGs were found in both preparations, but not in the corresponding normal splenic extracts. The amyloid‐associated GAGs of the extracts derived from one patient consisted of 60% dermatan sulphate and 40% n heparan sulphate whereas those obtained from the second spleen were 25% dermatan sulphate and 75% heparan sulphate. The heparan sulphate fraction occurred in the form of proteoglycans, whereas the dermatan sulphate apparently occurred as free GAG chains, resembling the data recently obtained from AA amyloid fibril extracts.
Highly Efficient Expression of Proteins Encoded by Recombinant Vaccinia Virus in Lymphocytes
Authors: ALONSO J. M , RODRIGUEZ J. , VIÑUELA E. , KROEMER G. , MARTÍNEZ‐A C.
Published: June 2006

Using a recombinant vaccinia virus (VV) that expresses E. coli β galactosidase (β‐Gal) to infect lymphocytes, we show that enzymometrically or immunologically detectable β‐Gal expression is less pronounced among T cells than among B cells, VV infection caused growth inhibition of B cells, but barely affected T‐cell proliferation in vitro. Moreover, the production of infectious viral particles was less pronounced in T lymphocytes. Kinetic studies revealed that after an initial dose‐dependent growth inhibition, T cells continued to proliferate without the doubling time being affected by VV infection. Nonetheless. The T cells do express proteins encoded by recombinant VV. such β‐Gal. or secrete soluble proteins such as interleukin‐4, though at a lower efficiency at the per cell level than B lymphocytes. In conclusion, the physiology of T cells appears lo be less perturbed by VV than that of B ceils, although the virus is capable of directing expression of recombinant genes to T lymphocytes.
Monoclonal Antibody Treatment (Anti‐CD4 and Anti‐Interleukin‐2 Receptor) combined with Cyclosporin A has a Positive but not Simple Dose‐Dependent Effect on Rat Renal Allograft Survival
Authors: STEINBROCHEL D. A. , KOCH C. , RISTENSEN T. K , KEMP E.
Published: June 2006

The use of monoclonal antibodies (MoAbs) in experimental and clinical organ transplantation is of increasing interest since this treatment seems to offer an opportunity for specific immunomodulation.

In a rat kidney allograft model. Cyclosporin A (CyA) treatment (12.5 mg/kg/d, day 0–14) was combined with murine anti‐rat CD4 (MRC OX‐38) and murine anti‐rat IL‐2R (MRC OX‐39) MoAbs at doses of i00 or 300μg;kg/d (day 0–7) and plasma concentrations of the murine MoAb were determined.

In both groups receiving combined treatment with CyA and MoAb, graft survival was prolonged to an average of 65 days, compared to a graft survival of 9–10 days in non‐treated recipients. Further, the data showed a beneficial effect of CyA + MoAb treatment versus CyA alone (graft survival 32 days). The threefold increased MoAb dose did not seem to improve graft survival or function. Treatment with OX‐38 + OX‐39 at a dose of 100 μg/kg/d each resulted in plasma levels of 280 ng/ml 14 days after transplantation. Corresponding values after the administration of 300 μg/kg/d were 1800 ng/ml in graft recipients as well as controls.

These findings indicate that the effect of MoAbs in complex organ transplantation models is not simply dose dependent and that in vitro assays are of limited value in predicting the effect of a given MoAb when used in vivo. The determination of MoAb plasma levels, however, may be a useful tool in defining optimal MoAb administration and to monitor therapeutically effective plasma levels.