bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences within Mono‐ and Dizygotic Twin‐Pairs in Spectrotypes and Clones of IgG2 Antibodies to Pneumococcal Polysaccharide Type 1 and C‐Polysaccharide After Vaccination
Authors: KONRADSEN H. B. , HAHN‐ZORIC M. , NAGAO A. T. , HANSON L. A.
Published: July 2006

Spectrotypes and clones of antibodies against pneumococcal capsular polysaccharide (Ps) type 1 and C‐polysaccharide (C‐Ps) were determined before and after immunization with a polyvalent pneumococcal Ps vaccine in 84 mono‐ or dizygotic twins. The method used was a micromodification of a rapid isoelectric focusing‐amnity (lEF‐affinity) immunoblot technique in agarose permitting characterization of isotype, light chain and Gm type. After vaccination the anti‐type 1 Ps + anti‐C‐Ps clones were different in 75% of the monozygotic and 79% of the dizygotic twins. The anti‐type 1 Ps clones differed among 72% of the monozygotic and 85% of the dizygotic twins (P > 0.05). Each twin had from zero to three clones producing IgG2 antibodies against type 1 Ps, A total of six different clones could be distinguished among all the twins. Vaccination enhanced the already actively secreting B‐cell clones in 56 twins and newly recruited clones in 11 of the 84 twins; six among the 48 mono‐ and five among the 36 dizygotic twins. These new clones differed among the twins. Spectrotypes varied between all twins within the pairs. The fact that all twins differed in spectrotype is due to post‐translational microheterogenity of the antibodies, events which are thus not genetically determined. The observation that even mono zygotic twins possessed and responded with different clones within the pairs indicates that the V‐region genes, which determine the final specificity of B cells, either differ from the original germ‐line V region genes, e. g. owing to hypermutations or junctional diversity, or the rearranged germ‐line genes occur accidentally although highly restricted.
Interleukin‐4 Induces In Vitro Migration in Naive B Cells Through Direct Mechanisms
Authors: CLINCHY B. , MOLLER G.
Published: July 2006

Interleukin‐4 (IL‐4) mediated locomotor responses by marine B cells in vitro were examined in this paper. The IL‐4 induced migration was found to act directly on purified, splenic B cells, without involvement of secondary mediators. It appeared that only a subpopulation of B cells was able to respond in migration assays. Flowcytometric analysis showed that the migrating cells had the characteristics of naive B cells: I‐A10, J 11dhi, IgDhi. They also displayed high expression of the adhesion molecule L‐selectin and made predominantly IgM antibodies. This is contrary to what has previously been observed regarding motile responses to chemotactic factors by T cells, which mostly affect memory or activated T cells. However, this is in accordance with other studies indicating that IL‐4 is a cytokine that exerts its effect mainly on resting B cells.
Expression of CD43 Epitopes on NK and T Cells
Authors: HØYSÆETER S. , JENSEN W. , POSSUM S.
Published: July 2006

CD43 epitope expression was studied with a panel of monoclonal antibodies (MoAbs) by immuno‐histochemistry on freeze sections of lymphoid tissues. The MoAb WEN3 stained most cells weakly in the T areas and scattered splenic red pulp cells strongly, whereas the other MoAbs strongly stained the majority of the cells in the T areas but gave variable staining patterns of cells in the non‐T areas. Flow cytometry on CD4 + and CD8 + T cells (T4 and T8 cells), freshly isolated NK cells and LAK cells showed distinct staining profiles for each cell type, with epitope expression patterns of T8 cells lying between those of T4 cells and NK/LAK cells. T8 cells were split by one of the MoAbs, the NK cells, but not LAK cells, were split by two other MoAbs.
Catabolism of the Murine IgGl Molecule: Evidence that Both CH2‐CH3 Domain Interfaces are Required for Persistence of IgGl in the Circulation of Mice
Authors: KIM J.‐K. , TSEN M.‐F. , GHETIE V. , WARD E. S.
Published: July 2006

Site‐directed mutagenesis of a recombinant Fc‐hinge fragment has previously been used to identify a region of the murine IgG 1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this ‘catabolic site’ has been further analysed using recombinant techniques. Fc‐hinge, CH2‐hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice. The CH2‐hinge has been analysed as both a monomer and dimer, and the dimer has a longer β phase half‐life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc‐hinge fragment comprising a heterodimer of one Fc‐hinge with the wild type (WT) IgGl sequence and a mutant Fc‐hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a β phase half‐life of 37.9 h and this is significantly shorter than that of the WT Fc‐hinge fragment (82.9 h). In contrast to the CH2‐hinge dimer, the CH3 domain is cleared rapidly (β phase half‐life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgGl molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.
Expression of CD28 on CD8+ and CD4+ Lymphocytes During HIV Infection
Authors: CARUSO A. , CANTALAMESSA A. , LICENZIATI S. , PERONI L. , PRATI E. , MARTINELLI F. , CANARIS A. D. , FOLGHERA S. , GORLA R. , BALSARI A. , CATTANEO R. , TURANO A.
Published: July 2006

CD28 interaction with B7 molecules, expressed on the membranes of antigen‐presenting cells, costimulates cytokine production, T‐cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4+ and CD8+ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28‐bearing CD4+ and CD8+ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non‐AIDS patients. At the same time, an increase in the absolute CD8+CD28− cell number (greater in stage A than in stage B and C subjects) was observed in HIV‐infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV‐infected patients.
Measurement of Allergen‐Specific IgD and Correlation with Allergen‐Specific IgE
Authors: ZHANG M. , NIEHUS J. , BRUNNÉE T. , KLEINE‐TEBBE J. , O'CONNOR A. , KUNKEL G.
Published: July 2006

A new method for the measurement of allergen‐specific IgD (as‐IgD) was developed by modifying the ImmunoCAP assay (Pharmacia), and amplification of the signal with a goat anti‐human/rabbit antigoat detection system. The assay was sensitive enough to measure as‐IgD in serum samples. The specificity of the assay was examined using inhibition tests with excess corresponding and non‐corresponding allergens. For the different allergens inhibition rates between 56% (house dust mite) and 88% (cat) could be achieved. Non‐corresponding allergens did not inhibit the as‐IgD binding. Total IgE and allergen‐specific IgE (as‐IgE) was measured using the ImmunoCAP system. Total IgD was measured using a sandwich ELISA. As‐IgD was measured in serum samples from 51 atopic and 2.1 non‐atopic subjects, and the correlation with as‐IgE was examined. As‐IgD was detected in both atopies and non‐atopies but at higher levels in atopies. As‐IgD against birch pollen and timothy pollen allergen was found to be increased in atopies with IgE directed against these allergens compared to atopies without IgE against these allergens (P <0.02 and P <0.03). As‐IgD against birch pollen allergen was higher in atopies with IgE specific to this allergen than in non‐atopies (P <0.02). In contrast to total IgE and total IgD, significant correlations were observed between as‐IgD and as‐IgE against timothy pollen (r= 0.34, P <0.04), birch pollen (r= 0.38, P <0.05) and cat dander allergen (r= 0.52, P <0.01). The observed correlations between as‐IgD and IgE suggest that IgD and IgE may be similarly regulated, and thus the measurement of as‐IgD may give further insight into the regulation of IgE.
The Antibody MY4 Recognizes CD14 on Porcine Monocytes and Macrophages
Authors: ZIEGLER‐HEITBROCK H. W. L. , APPL B. , KÄFFERLEIN E. , LÖFFLER T. , JAHN‐HENNINGER H. , GUTENSOHN W. , NORES J. R. , MCCULLOUGH K. , PASSLICK B. , LABETA M. O. , IZBICKI J.
Published: July 2006

Several monoclonal antibodies directed against the human CD14 antigen have been established. We now report that the antibody My4, but not LeuM3, reacts with porcine monocytes. Among porcine peripheral blood mononuclear cells (PBMC), 14.6% of the cells stain with the CD14 antibody My4, which is similar to the percentage obtained with the antiporcine monocyte antibody 74‐22‐15. Two‐colour immunofluorescence reveals that My4 and 74‐22‐15 antigens are coexpressed on the same cells, and cell sorter‐purified My4+ cells exhibit the morphology of monocytes. Whole blood analysis (which also shows staining of granulocytes) reveals that the average percentage of My4+ monocytes amongst all leucocytes is 5.8% with 580 cells/μl. Furthermore, porcine peritoneal macrophages (PM) and alveolar macrophages (AM), both stain for My4, with a four‐fold lower level on AM. Treatment of cells with phosphatidylinositol‐specific phospholipase C decreases My4 staining, but does not affect staining with antibody 74‐22‐15. Immunoprecipitation with the My4 antibody from surface labelled pig mono‐nuclear cells demonstrates a 54 kDa band similar to human CD14, and Western blotting with pig serum demonstrates two bands similar to the alpha and beta forms of human soluble CD14. Finally, the My4 antibody is capable of blocking lipopolysaccharide‐ (LPS)‐induced interleukin‐6 production in isolated PBMC. These data show that the My4 antibody recognizes genuine CD14 on porcine monocytes and macrophages.
An In Vitro Model for the Expansion of Vγ9δ2 T Lymphocytes During Development
Authors: WILHELM M. , TONY H.‐P.
Published: July 2006

The 7/γ/δTlymphocytes represent a minority of T lymphocytes in human peripheral blood. Although there have been reports of reactivity against (myco‐) bacterial antigens and heat shock proteins, their function and antigen specificity remain ill defined. The biological role of γ/δ T cells has been related to functions within the ‘first line of defense’. Similar to γ/δ T lymphocytes in the T‐cell compartment, CD5 positive B cells represent a small subset of B lymphocytes, which is thought to be involved in the maintenance of natural immunity and autoimmunity. We provide evidence for the cooperation of γ/δ T cells and CD5 positive B cells in the proliferative response of γ/δ T cells to bacterial antigens. Our data indicate a strong proliferation of Vγ9δ2 T cells in response to gram‐negative bacteria, which is dependent upon the presence of CD5 positive B‐CLL or activated normal B lymphocytes. The selective stimulation of the Vγ9δ2 subpopulation by gram‐negative bacteria is also confirmed by analysis of different γ/δ T‐cell clones. The interaction of γ/δ T cells with activated B cells and gram‐negative bacteria may prove to be a useful model similar to the expansion of the Vγ9δ2 subpopulation during development. In addition, our in vitro system should provide new insights in the interaction of CLL B cells with the immune system and the antigens recognized by γ/δ T cells.
Human Th0‐type T Helper‐Cell Clone Supports Antigen‐Specific Immunoglobulin Production in Scid/beige‐hu Mice
Authors: CHIN L.‐T. , IFVERSEN P. , KRISTENSSON K. , WAHREN B. , CARLSSON R. , BORREBAECK C. A. K.
Published: July 2006

Tetanus toxoid‐specific T cells have been generated from human splenic lymphocytes by an initial 6‐day stimulation period with antigen, followed by a proliferation period with recombinant IL‐2 and human feeder cells. Proliferating T cells were subsequently cloned by limiting dilution. A human T‐cell clone that was functionally characterized showed: (i) a specific proliferative response to tetanus toxoid in the presence of autotogous Epstein—Barr virus (EBV)‐transformed lymphoblastoid cells; (ii) a phenotype characteristic for the helper/inducer CD4+/CD8−/CD45RO+ T cells, and (iii) a lymphokine profile, as determined by mRNA analysis, representative of Th0‐like human CD4+ T helper cells. This tetanus toxoid‐specific T‐cell clone which showed antigen‐dependent helper activity for antibody production by autologous B cells in vitro could also provide T‐cell help to antigen‐specific human B cells transplanted into severe combined immunodeficiency (scid/beige) mice.
I. Immunostimulatory Effect of Thalidomide in Normal C57BL/6 Mice is Compatible with Stimulation of a Highly Connected Central Immune System
Authors: ARALA‐CHAVES M. , VILANOVA M. , RIBEIRO A. , PINTO J.
Published: July 2006

Although thalidomide has been used with success in the treatment of increasing numbers of autoimmune diseases, the therapeutic effects have not been satisfactorily explained so far. We describe here some findings that may contribute to a better understanding of the immunomodulatory effects of this drug. Several immunological changes were observed after treating C57BL/6 mice with 3 mg of thalidomide. The numbers of natural IgM PFC against sheep red blood cells were increased in the spleen, and occasionally a dramatic oscillatory increase in the numbers of non‐specific splenic IgM and IgG PFC was observed in these mice. However, these oscillatory increases were progressively lower, after two and three treatments with thalidomide at 20‐day intervals. Furthermore, the absolute numbers of splenic CD5+ B and CD5 B lymphocytes were increased whereas depletion of CD4+ CD8+ cells in the thymus and of lymphoid cells in the bone marrow was seen after a single treatment with 3 mg of thalidomide. Taken together, these results suggest that thalidomide stimulates both peripheral and central immune systems and consequently enhances the connectivity of the central immune system.
II. The Effects of Thalidomide Treatment on Autoimmune‐Prone NZB and MRL Mice are Consistent with Stimulation of the Central Immune System
Authors: VILANOVA M. , RIBEIRO A. , CARNEIRO J. , ARALA‐CHAVES M.
Published: July 2006

We describe here some immunomodulatory effects of thalidomide on autoimmune‐prone mice. The highly increased synthesis of splenic IgM in NZB mice, of splenic and lymph node IgG of different subclasses in MRL/n mice, and of splenic and lymph node IgGl in MRL/lpr mice was markedly inhibited by thalidomide treatment. After a single treatment with 3mg of thalidomide, the following changes were observed in NZB mice: (i) an initial decrease in the numbers of large CD5+μhigh, and in the numbers of total CD5+μ−, CD5−μhigh, CD5+μhigh lymphocyte populations of the pleural cavity followed by a late increase in the numbers of large cells of the three cell populations; (ii) a consistent increase in the numbers of a CD5lowμlow pleural lymphoid population; (iii) a consistent reduction in the numbers of splenic large CD5+ B cells and an oscillatory increase in the number of cells with CD5− phenotype; (iv) a late reduction in the numbers of splenic total CD5+ B cells. These results are consistent with the notion that thalidomide controls a disease‐associated expansion of B cells in autoimmune prone mouse strains through a stimulatory effect of the drug on the immune system.
Identification of the Site of Uptake of the E. coli Heat‐labile Enterotoxin, LTB
Authors: LINDNER J. , GECZY A. F. , RUSSELL‐JONES G. J.
Published: July 2006

The results of this study demonstrate that the B subunit of the E. coli heat labile toxin (LTB) binds to the brush border of intestinal epithelial cells in a highly specific, lectin‐like manner. Uptake of LTB and transcytosis to the basolateral side of the enterocytes can be observed within 1 h after feeding, and occurs through both the villous epithelial cells and the epithelial cells overlying lymphoid follicles and Peyer's patches. Binding and uptake most probably occur via receptor‐mediated endocytosis, with GM1 ganglioside and galactoproteins on the enterocyte cell surface acting as specific ligands to which the LTB binds. Cell EL1SA data, together with the observed distribution of immunocompetent cells and the localization of LTB binding, suggest that LTB which is taken up by the villous enterocytes enters the circulation and subsequently generates an IgG immune response in the spleen. At the same time. LTB which is taken up via the patch associated epithelium generates a local IgG and IgA immune response within the Peyer's patches and intestinal lymphoid follicles.
The Influence of Branched Polypeptide Carriers on the Immunogenicity of Predicted Epitopes of HSV‐1 Glycoprotein D
Authors: HILBERT Á. , HUDECZ F. , MEZÔ G. , MUCSI I. , KAJTÁR J. , KURUCZ I. , GERGELY J. , RAJNAVÖLGYI É.
Published: July 2006

To investigate the role of synthetic polypeptide carriers in inducing an epitope‐specific immune response relevant for vaccine design, peptides comprising two distinct regions of herpes simplex virus type I glycoprotein D (1–23 and 273–284) have been conjugated to the branched polypeptides with polylysine backbone, poly[L‐Lys‐(DL‐Alam)] (AK), or poly[L‐Lys‐(LeUi‐DL‐Alam)] (LAK) and to keyhole limpet haemocyanin (KLH). The magnitude, fine specificity and isotype distribution of the conjugate‐, peptide and carrier‐specific antibody responses were characterized in immunized BALB/c and CBA mice. Conjugates containing the polypeptide carrier AK were the most effective in inducing HSV gD‐peptide specific antibody responses while KLH peptide conjugates resulted in conjugate‐specific antibody responses without measurable peptide specificity. The efficacy of AK‐peptide conjugates was verified by the dominant appearance of peptide‐specific antibodies belonging to functionally efficient IgG isotopes, accompanied by low levels of carrier specific antibody responses. Preimmunization of BALB/ or CBA mice with AK conjugates comprising the 1–23 or 276–284 HSV peptides resulted in prolonged survival of animals infected with a lethal dose of infectious HSV‐1. The potency of these conjugates in eliciting a protective immune response shows a close correlation with the relative levels of conjugate‐induced virus specific antibodies and the neutralizing activity of sera as measured in preimmunized survivors.
IgG Subclass‐Associated Differences in Anti‐Schistosomal Antibody Specificity
Authors: Evengård B. , WOLOWCZUK I. , MARGUERITE M. , HAMMARSTRÖM L. , SMITH E. , AURIAULT C.
Published: July 2006

Consecutive sera from before and after treatment were collected from 23 patients with a Schistosoma mansoni infection, seven of whom had an early infection. All sera were analysed for IgGl, IgG3 and IgG4 antibody activity against three peptides from the protein Sm 28 GST (24–43, 115–131 and 140–153 aa). In addition, sera from 14 patients, four with an early infection and 10 patients with a chronic infection, were analysed for IgG and IgA antibody activity using seven peptides derived from the protein Sm 28 GST. This molecule has previously demonstrated protective activity against infection in various experimental models.

The results are indicative of a subclass‐related epitope specificity of the antibodies. Moreover, reactivity to one of the peptides (158–175 aa) was significantly associated with a chronic infectious status.
T Cells from Children with IDDM are Sensitized to Bovine Serum Albumin
Authors: CHEUNG R. , KARJALAINEN J. , VANDERMEULEN J. , SINGAL D. P. , DOSCH H.‐M.
Published: July 2006

Epidemiological and experimental evidence suggested that denial of dietary cow milk protein early in life protects genetically susceptible children and animals from insulin‐dependent diabetes (IDDM). Bovine serum albumin (BSA) was proposed as a candidate milk‐borne mimicry antigen responsible for the diabetogenic cow milk effect. Elevated anti‐BSA antibodies have been observed in patients and diabetic rodents, and these antibodies precipitate p69 from islet cell lysates. IDDM is a T ceil mediated disorder but efforts to detect BSA‐specific T cells in diabetic children have so far failed. We describe here a culture system which allowed the detection of BSA‐specific T cells and we mapped this response to the ABBOS peptide (pre‐BSA position 152–169) previously identified as a possible mimicry epitope. ABBOS sensitized T ceils were found in 28/31 children with recent onset TDDM but not in non‐diabetic controls nor in children with SLE or JRA. T cell proliferative responses declined within the first few years of diabetes diagnosis. Although no effector cell role for BSA/ABBOS specific T lymphocytes has been demonstrated, the presence of BSA peptide‐specific T cells strengthens the postulated link between a cow milk protein and IDDM.
IgG Humoral Response Against the Antigen 85 Complex Homologues in Leprosy
Authors: DROWART A. , HUYGEN K. , LAUNOIS P. , JANN E. , NYABENDA J. , VAN VOOREN J. P.
Published: July 2006

Antigen 85 complex is the major protein component present in M. bovis BCG culture filtrate (CF). It consists of a family of three proteins: 85A, 85B and 85C. Combining isoelectric focusing and Western blot analysis, we have previously identified different antigenically related proteins present in the CF of other mycobacteria (M. tuberculosis, M. kansasii, M. avium, M. gordonae, M. fortuitum and M. plilei) using monoclonal antibodies (MoAbs) directed against the antigen 85 complex of M. bovis BCG.

Humoral immune response directed against these cross‐reactive homologues was analysed in sera from 20 patients with multibacillary leprosy (BL/LL), from 20 patients with paucibacillary leprosy (BT/ TT) and from 15 healthy leprosy contacts.

All the antigen 85 homologues identified in the seven CFs by MoAbs were also recognized by IgG present in sera from multibacillary leprosy patients, but not or very faintly in sera from paucibacillary leprosy patients or from healthy subjects.

These results suggest that some of the M. leprae epitopes inducing a significant humoral response in multibacillary leprosy are common to the various 85 antigenically related proteins present in all mycobacterial species.
Direct Evidence for SAA Deposition in Tissues During Murine Amyloidogenesis
Authors: YAKAR S. , KAPLAN B. , LIVNEH A. , MARTIN B. , MIURA K. , ALI‐KHAN Z. , SHTRASBURG S. , PRAS M.
Published: July 2006

To study the mechanism of amyloid deposition, the nature of amyloid proteins formed in experimental murine amyloidosis, was examined. Spleen specimens, 15–60mg, were homogenized and extracted using aqueous acidic acetonitrile, in a recently developed procedure, making it possible to obtain amyloid proteins from minute amounts of tissue. The extracted material, 1.5–4mg, was analysed by Western blotting and ELISA using antibodies recognizing differentially proteins AA and SAA. Two immunoreactive proteins of 8 and 12 KDa were isolated and subjected to amino acid analysis and N‐terminal sequence determination. The results of immunochemical and chemical examination showed that the 8 and 12 KDa proteins represented proteins AA and SAA, respectively.

The data obtained provide new direct evidence for SAA deposition in tissues during murine amyloidogenesis.
Non‐Diabetogenic Insulitis in NOD↔BIO. GD Allophenic Mice in Spite of Permissive Class I MHC Antigens
Authors: COLUCCI F. , DAHL U. , O'REILLY L. , COOKE A. , CHANDLER P. , SIMPSON E. , MATSUNAGA T.
Published: July 2006

Allophenic mice (embryo aggregation mouse chimeras) enable us to dissect the process of spontaneous autoimmunity under physiological conditions. Our previous experiments showed that the autoimmune process in allophenic mice of the NOD↔C57Bl/6 strain combination does not progress from insulitis to diabetes. One possible explanation for this protection is that H‐2 Kd‐restricted CD8+T cells kill only NOD ß cells (Kd, Db) in the chimeric islets, while the B6ß cells (Kb, Db) are spared from destruction. To test this hypothesis we analysed 22 NOD↔B10. GD chimeras in which the class I MHC are shared by both parental strains. Therefore all the ß cells in these chimeras express H‐2 Kdmolecules. Ten allophenic mice were killed at 7 weeks and studied for early pathology. No evidence for intra‐islet infiltration was obtained at this age, suggesting that the autoimmune process in NOD↔B10. GD chimeras is slower than in NOD mice. Twelve chimeras were followed up for 1 year for disease development and all failed to progress to full‐blown diabetes, despite the occurrence of intra‐insulitis in six out of 12 mice. The lack of disease in NOD↔B10. GD chimeras demonstrates that class I MHC chimerism does not account for diabetes resistance in NOD‐allophenic mice.
Characterization of a Functional Null Cell Line Derived from (NZB × NZW)F1 Mice
Authors: IWASAKI T. , HAMANO T. , AIZAWA K. , KOBAYASHI K. , KAKISHITA E.
Published: July 2006

We attempted to establish a null cell line from NZBxNZW(B/W)Fl mice in order to investigate a regulatory role of null cells during polyclonal B cell activation in autoimmune diseases. NB2.2, a representative subclone of resulting null cell lines, was maintained in long‐term tissue culture with 10% mouse ConA supernatant (MCAS). Interestingly, the cell free supernatant of the NB2.2 cells (NB‐CFS) showed marked synergistic effects on IgM secretion by B cells induced by IL‐5. In addition, NB‐CFS had the ability to augment the production of autoantibodies against bromelain‐treated mouse red blood cells (BrMRBC) and single‐stranded DNA (ssDNA) by B cells induced by IL‐5. To determine whether NB2.2 cells induce polyclonal B cell activation and autoantibody generation in vivo, BALB/c mice were injected with NB2.2 cells. The results showed that the level of anti‐ssDNA antibodies in sera of BALB/c mice injected with NB2.2 cells was significantly higher than that of control BALB/c mice injected with FDC‐P2 cells. In addition, splenic B cells from mice injected with NB2.2 cells significantly proliferated in vitro in response to IL‐4 and IL‐5, and produced anti‐ssDNA antibodies in the presence of IL‐5. These results suggest that NB2.2, a null cell line established from B/WF1 mice, produces mediators capable of promoting polyclonal B cell activation and inducing autoantibody secretion, and that this kind of null cell may play an important role in the pathogenesis of autoimmune diseases.
Effects of Human Calprotectin (L1) on In Vitro Immunoglobulin Synthesis
Authors: BRUN J. G. , ULVESTAD E. , FAGERHOL M. K. , JONSSON R.
Published: July 2006

Calprotectin (LI) is a major cytoplasmic protein of neutrophilic granulocytes and monocytes/macrophages which is released from leucocytes during activation or cell death. Apart from in vitro antimicrobial and antiproliferative activity little is known about the biological function of the protein. Since previous investigations have shown that Calprotectin plasma levels are elevated in various inflammatory rheumatic diseases, we wanted to investigate if Calprotectin has an effect on immune cell functions. Peripheral blood mononuclear cells, either unstimulated or polyclonally stimulated with mitogen, were incubated with Calprotectin and effects were assessed by enumeration of immunoglobulin secreting cells (ELISPOT).

The results indicate that incubation with high concentrations of Calprotectin (> 64 μ/ml) inhibit the production of the three classes of immunoglobulins investigated (IgG, IgM and IgA), both for mitogen stimulated and unstimulated lymphocytes. Except for the highest concentration of Calprotectin (500 μ/ml), it seems plausible that the observed inhibitory effect of Calprotectin on Ig production is not a result of a direct toxic effect of Calprotectin on B lymphocytes. Altogether, these effects of high Calprotectin levels might be of importance in the immunoregulation of inflammatory conditions
Characteristics of Cytotoxic T Lymphocytes Directed to Influenza Virus Haemagglutinin Elicited by Immunization with Muramyldipeptide‐Influenza Liposome Vaccine
Authors: HNUMA H. , NEROME K. , YOSHIOKA Y. , OKINAGA K.
Published: July 2006

We examined the characterization of the antiviral T lymphocytes elicited by immunization with a novel liposome vaccine (MDP‐virosome) constructed with synthetic muramyldipeptide:[6‐0‐(2‐tetradecylhexadecanoyl)‐N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine], cholesterol, influenza virus haemagglutinin and neuraminidase. The haemagglutinin glycoprotein first appeared to induce a significant subtypespecific cytotoxic activity through its arrangement on the inner and outer surfaces of the MDP‐virosome. Splenocytes of BALB/c mice immunized with the virosome vaccine containing H3 haemagglutinin and N2 neuraminidase from human Hong Kong virus markedly lysed H3N2 virusinfected target cells, but not those infected with virus possessing a different subtype such as H1N1 surface antigens. Exposure of these splenic lymphocytes to virus antigen in vitro further enhanced their cytotoxic activity. The cytotoxic lymphocytes generated by the MDP‐virosome vaccine expressed Thy 1 and CD4 antigens on their cell surface, and these activities were restricted by class II histocompatibility gene products. The marked reduction of pulmonary virus litres in infected mice caused by transferred immune spleen cells suggested that the MDP‐virosome vaccination is able to protect against influenza virus infection through enhanced cellular immune responses.
Subpopulations of T Lymphocytes in the Peripheral Blood, Dermal Lesions and Lymph Nodes of Post Kala‐azar Dermal Leishmaniasis Patients
Authors: GHOSH M. K. , NANDY A. , ADDY M. , MAITRA T. K. , GHOSE A. C.
Published: July 2006

Distribution of different subpopulations of T cells in the dermal lesions, lymph nodes and peripheral blood of post kala‐azar dermal leishmaniasis (PKADL) patients was studied by using appropriate phenotypic markers for CD2+, CD4+ and CD8+ cells. Histopathological studies of skin lesions showed marginal to massive infiltration of mononuclear cells depending upon the duration of illness and type of lesions. Thus, while the hypopigmented patches were represented by small focal collections of lymphocytes with scanty parasites in the dermis, these were replaced at the nodular stage with massive granulomas consisting of lymphocytes, plasma cells and histiocytes with numerous amastigotes. The involvement of CD4+ and CD8+ cell types in these lesions also showed a gradual change from the appearance of a few cells of both the phenotypes in early hypopigmented type to massive accumulation of cells, primarily of CD8+ phenotype, in the granuloma of nodular type. However, the observed preponderance of CD8+ cells at the lesion site of chronic PKADL patients is in contrast to their peripheral blood CD4+/CD8+ cell ratio (1.9:1) which remained within the normal limits. Similar studies of lymph nodes from PKADL patients with lymphadenopathy revealed infiltration of the cortical areas by T cells which were more of CD8+ than CD4+ phenotypes. All these results document the involvement of CD8+ cells in leishmanoid lesions. Thus, it is likely that these cells, in association with appropriate subpopulations of CD4+ cells, play a profound role in the evolution of dermal pathology in PKADL.
Enhancement of Infectivity of a Non‐Syncytium Inducing HIV‐1 by sCD4 and by Human Antibodies that Neutralize Syncytium Inducing HIV‐1
Authors: SCHUTTEN M. , ANDEWEG A. C. , BOSCH M. L. , OSTERHAUS A. D. M. E.
Published: July 2006

Enhancement of virus infectivity after sCD4 treatment has been documented for SIVagm and HIV‐2. It has been suggested that a similar phenomenon may play a role in HIV‐1 infection. In the present study we have analysed biological activities of virus neutralizing polyclonal and monoclonal human antibodies and of sCD4, towards HIV‐1 chimeras with envelope proteins derived from one donor, which display different biological phenotypes. The antibodies, which recognize the V3 and/or the CD4 binding domains of the glycoproteins of these viruses and also sCD4 showed different levels of virus neutralizing activity toward the syncytium inducing HIV‐1 strains. In contrast, they all dramatically enhanced the infectivity of an HIV‐1 chimera with an envelope glycoprotein displaying the non‐syncytium‐inducing phenotype. Given the relatively conserved nature of non‐syncytium‐inducing HIV‐1 surface glycoproteins early after infection, these data suggest a major role for antibody mediated enhancement of virus infectivity in the early pathogenesis of HIV‐1 infection.
Cytokines and Murine Autoimmune Encephalomyelitis: Inhibition or Enhancement of Disease with Antibodies to Select Cytokines, or by Delivery of Exogenous Cytokines Using a Recombinant Vaccinia Virus System
Authors: WILLENBORG D. O. , FORDHAM S. A. , COWDEN W. B. , RAMSHAW I. A.
Published: July 2006

To examine the complex role of cytokines in the pathogenesis of actively induced murine EAE we measured the levels of a number of cytokines (IL‐6, IFN7 and TNF) in the spinal cord and CSF of mice with active experimental autoimmune encephalomyelitis (EAE) and found them all to be elevated. We next treated mice with antibodies to these three cytokines, which were over expressed in the CNS, to determine if they would alter disease and found the following: anti‐IL‐6 had no significant effect on disease, anti‐IFNγ exacerbated disease, and anti‐TNF either enhanced, had no effect or inhibited EAE depending on the antibody used. We then treated mice with exogenous cytokines, delivered using a recombinant vaccinia virus system, and found that the IL‐6 and TNF virus constructs inhibited EAE whereas the IFN1β construct had no effect on disease. Other cytokine recombinant viruses were also tested and it was found that the IL‐1β, IL‐2 and IL‐10 viruses inhibited EAE while an IL‐4 virus either had no effect or enhanced disease. We do not know the mechanism of action of the various cytokines in this system, but irrespective of the mechanism(s), this work clearly demonstrates that delivery of select cytokines using recombinant virus‐cytokine constructs can provide a powerful means of downregulating experimental organ‐specific autoimmune disease.
Freshly Isolated Tumour‐Infiltrating T‐Lymphocytes have a High Cytotoxic Potential, as Measured by their Ability to Induce Apoptosis in the Target Cell
Authors: ØSTENSTAD B. , SIOUD M. , SCHLICHTING E. , LEA T. , HARBOE M.
Published: July 2006

To test if freshly isolated tumour‐infiltrating lymphocytes (TIL) can induce apoptosis in a target cell, we have combined two previously described methods. Because TIL predominantly are T‐lymphocytes. we have applied a redirected approach. When the target cells that express anti‐human‐CD3 monoclonal antibodies in their membranes bind to the T cell receptor‐associated CD3‐complex, signals are generated, which activate T cell effector mechanisms. This approach circumvents problems with MHC‐restriction and allows for functional testing of all T cells, irrespective of their clonal specificity.

In order to assay for induction of DNA fragmentation, we have labelled the target cell nuclei with [3H]thymidine. Upon harvesting fragmented DNA are washed away. Electrophoretic analysis of the fragmented DNA demonstrated the characteristic ‘ladder’ pattern, consistent with apoptosis.

This rapid and simple assay monitors the capacity of different T cells to induce apoptosis in the target cell. It depends on intercellular interactions and clearly discriminates between different T cell subsets. With this assay we demonstrate the functional integrity of the cytotoxic effector arm of freshly isolated TIL.
Increased Levels of Antibodies to IFN‐γ in Human and Experimental African Trypanosomiasis
Authors: BONFANTI C. , CARUSO A. , BAKHIET M. , OLSSON T. , TURANO A. , KRISTENSSONP K.
Published: July 2006

In African trypanosomiasis the occurrence of antibodies to interferon‐γ (IFN‐γ) was studied in both humans and experimental rats. Sera from patients infected with Trypanosoma brucei gambiense showed increased levels of antibodies to IFN‐γ as compared with controls from the same regions in Africa. In Sprague‐Dawley rats infected with Trypanosoma brucei brucei an early appearance of IFN‐γ‐producing spleen cells was observed, followed by an increase in levels of antibodies against IFN‐γ in the sera. Previously, IFN‐γ has been found to play a crucial role in trypanosome infections in rats by promoting proliferation of Trypanosoma brucei brucei. The appearance of antibodies to IFN‐γ in humans, as in rats, indicates that this cytokine is produced also in the human infection. Its parasitic growthstimulating and pathophysiological effects on the organism may be reduced by the antibodies.
Alteration of the Immune Response During Cancer Development and Prevention by Administration of a Mycobacterial Antigen
Authors: MAES H. , TAPER H. , COCITO C.
Published: July 2006

It has been shown previously that A60, an antigen complex of Mycobacterium hovis BCG, triggers humoral and cellular immune reactions in vivo and lymphocyte‐dependent macrophage activation in vitro. In the present work, the ability of A60 to prevent murine tumour development, in conjunction or not with irradiated isologous cancer cells, was explored with Taper liver tumour (TLT), a mammar‐y‐derived neoplasm (EMT6), and Lewis lung carcinoma (3LL). Repeated injections of A60 prior to challenge reduced the incidence of EMT6 and 3LL solid tumours and increased life span. This effect was enhanced by simultaneous administration of 7‐irradiated cancer cells (80–100% suppression of EMT6 and 3LL tumour growth). In mice developing or rejecting tumours, the status of humoral and cellular immunity was evaluated by A60‐based immunoassays. Tumor development was accompanied by a rapid decrease of both anti‐A60 IgG titre in blood and A60‐triggered delayed hypersensitivity reactions. Moreover, A60‐induced T lymphocyte proliferation and macrophage‐dependent autologous cancer cell cytolysis declined progressively during the course of tumour growth. In case of successful immuno‐therapy, a pattern similar to that of unchallenged controls was observed. Our results suggest that A60 promotes cancer rejection via tumour infiltration by lymphocytes and macrophages activated by A60‐specific T lymphocytes. An increased processing of tumour‐specific antigens and activation of tumour‐infiltrating lymphocytes is induced by administration of irradiated cancer cells in conjunction with A60.
Requirement of CD4+ Lymphocytes in IL‐2‐Stimulated NK Cell Proliferation
Authors: KOVANEN P. E. , KNUUTILA S. , TIMONEN T.
Published: July 2006

Growth requirements of human natural killer cells in IL‐2‐supplemented cultures were studied, NK cell proliferation was monitored by the MAC (morphology antibody chromosomes) technique and subset specific cell cycle analysis, which both enable direct determination of cell growth in specific lymphocyte subsets among heterogeneous lymphocyte populations. Our results show that even in the presence of saturating concentrations of IL‐2, the proliferative capacity of purified CD16+ cells is quite low, but can be stimulated in a dose dependent manner by CD4+ cells. CD4+ cells could partially be replaced by IL‐4 but not by various other commercially available cytokines. These results provide further evidence of the requirement of accessory stimuli in NK cell proliferation, and support the interpretation that NK cells have a direct regulatory role in specific T cell responses.
Monocyte Activation by Tumour Cells: a Role for Carbohydrate Structures Associated with CD2
Authors: PUTZ E. F. , MÄNNEL D. N.
Published: July 2006

Monocytes/macrophages can kill tumour cells and mediate tumour‐destructive host responses e. g. by releasing tumour necrosis factor‐γ (TNF‐γ). The underlying mechanisms of tumour cell recognition, however, are not clear. Previous work in our laboratory suggested that carbohydrate moieties associated with the T cell adhesion molecule CD2 of Jurkat cells induce TNF‐γ secretion by human monocytes. In this study we present data indicating that the stimulatory capacity for TNF‐γ secretion is confined to carbohydrate moieties of tumour cell CD2. Irradiated resting peripheral T cells did not display stimulatory activity in contrast to irradiated Jurkat cells although surface expression of CD2 was similar. Activated T cells, however, induced TNF‐γ production by monocytes via a CD2‐independent mechanism. Only affinity purified CD2 prepared from Jurkat cells but not from non‐transformed T cells activated monocytes to secrete TNF‐γ. This activation process was blocked by anti‐CD2 antibodies. Neuraminidase and PNGaseF treatment of isolated CD2 inhibited the stimulatory capacity whereas pronase treatment did not. These data suggest that carbohydrate moieties containing siaiic acid mediate stimulation of monocytes. Taken together, these results indicate a role for glycosylation patterns typical of tumour cells in the recognition process of tumour cells by monocytes/ macrophages.
Peptide Mapping of Bovine T‐Cell Epitopes for the 38 kDa Tuberculosis Antigen
Authors: POLLOCK J. M. , DOUGLAS A. J. , MACKIE D. P. , NEILL S. D.
Published: July 2006

Mycobacterium bovis infection in cattle continues to be a problem in several regions, partly due to inadequate diagnostic tests. The aim of this study was to use an experimental model of the natural disease to identify T‐cell epitopes from the mycobacterial 38 kDa antigen as potentially specific diagnostic reagents. A panel of overlapping synthetic peptides (16‐mers with a five‐residue overlap) were produced from the published amino acid sequence. It was found that peripheral blood lymphocytes from at least three of four experimentally infected animals, which were considered to be in either Th1‐ or Thl/Th2‐dominated stages of anti‐mycobacterial immunity, proliferated in response to five epitopes (residues 1–27, 88–107, 122–138, 243–260 and 307–328). However, in vitro production of IFN‐γ was detected only in response to epitope 122–138, indicating a role in protective immunity. The peptides were not recognized by control, uninfected animals, but all epitopes showed various degrees of recognition by animals which were field reactors to intradermal tuberculin testing. Furthermore, epitopes 1–27, 88–107 and 122–138 were recognized by four breeds of cattle and by animals from separate herds, suggesting genetic permissiveness in recognition which would be essential in the development of a diagnostic test.
Cathepsin B Generates the Most Common Form of Amyloid A (76 Residues) as a Degradation Product from Serum Amyloid A
Authors: YAMADA T. , LIEPNIEKS J. J. , KLUVE‐BECKERMAN B. , BENSON M. D.
Published: July 2006

Amyloid A protein (AA), the chief constituent of reactive amyloid deposits, is derived from serum amyloid A (SAA) and most commonly corresponds to the amino‐terminal 76 residues (AA76). Digestion of recombinant human SAAl with a lysosomal thiol protease, cathepsin B. and analysis of the products by SDS‐PAGE and amino‐terminal sequencing revealed that AA76 was generated as a minor and transient degradation product. Digestion with neutrophil eiastase generated intermediates different from AA76. This finding suggests that cathepsin B may play an important role in amyloid fibrilogenesis by converting SAA to AA.
Cell‐Cycle Kinetics of Proliferating Mouse B Lymphocytes In Vitro
Authors: KÄLLBERG E. , LEANDERSON T.
Published: July 2006

Resting mouse B lymphocytes were stimulated in vitro with Upopoiysaccharide, Sepharose‐coupled anti‐γ antibodies or a combination of the two. B lymphocytes stimulated with anti‐γ entered the cell‐cycle with more rapid kinetics and at a higher frequency than did the corresponding cell population stimulated with lipopolysaccharide. Using cell cycle analysis after DNA staining combined with an M phase block, the cell‐cycle kinetics of in vitro cultured B‐lymphocytes was studied. The labelling index of lipopoly saccharide stimulated B lymphocytes was 60% while that for anti‐γ Sepharose stimulated cells was 85%. The generation time of the actively cycling population from both types of cultures was constant and was of the order of 18 h. Thus, the fraction of B lymphocytes induced to proliferate in vitro varies depending on the stimulus, while the growth kinetics of the actively proliferating populations are remarkably constant.
Regional Specialization of Intraepithelial T Cells in the Murine Small and Large Intestine
Authors: BOLL G. , RUDOLPHI A. , SPIEβ S. , REIMANN J.
Published: July 2006

We investigated intraepithelial T cells from the small intestine, SI (jejunum, ileum) and the large intestine, LI (colon) of euthymic (BALB/c, H–2d; C. B–17 +/+, H–2d; C57BL/6, H–2b) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. From individual euthymic and athymic mice, 7 × 106 intraepithelial lymphocytes (IEL) per mouse were isolated from the SI. Ten–fold lower numbers of IEL were obtained from the LI epithelium (4 × 105 IEL per mouse). Thymus–dependent and ‐independent T cells represented > 80% of SI–IEL but the fraction of T cells was reduced from 20% to 40% in LI–IEL. In euthymic mice, αβ T cells predominated in SI–IEL and in particular in LI–IEL populations, while SI–IEL and LI‐IEL populations of athymic mice contained predominantly αβ T cells. The intraepithelial T cell subset distribution was different in SI versus LI: mainly CD8+ T cells were present in the SI, but a large CD4+ T cell subset was present in the LI.‘Double positive’ CD4+ CD8α+ T cells were present mainly in the SI epithelium but were rare in the LI epithelium. In euthymic as well as athymic mice, T cells expressing the homodimeric CD8αβ isoform predominated in the SI epithelium, while T cells expressing the heterodimeric CD8αβ isoform predominated in the LI epithelium. LI–derived TCRα+ IEL displayed the CD2+ CD28+ LPAM–1/2− M290+ phenotype, and a fraction of them expressed the L–selectin LECAM–1. In contrast, a large fraction of TCRα+ SI‐IEL was CD2− CD28− LPAM–1/2− M290+ and LECAM–1−. RAG–1/2 expression was detectable by RT–PCR in IEL from the SI but not the LI. Striking differences in phenotype were thus apparent between thymus–dependent and thymus–independent T cells in the epithelial layer of the jejunum/ileum and the colon of the mouse.
Three‐Colour Flow Cytometric Immunophenotyping in HIV‐Patients; Comparison to Dual‐Colour Protocols
Authors: LILLEVANG S. T. , SPROGØE‐JAKOBSEN U. , SIMONSEN B. , KRISTENSEN T.
Published: July 2006

Flow cytometric measurement of circulating CD4+ lymphocytes is important in the evaluation of disease progression in HIV–infected patients. Development of dyes that can be exited at 488 nm and have emission maximum in the far red area has made three–colour protocols, together with fluorescein isothiocyanate (FITC) and R–phycoerythrin (PE), possible in most clinical flow cytometers. We report here the comparison of a two–tube, three–colour protocol (including CD45/CD4/CD3 and CD8/CD4/CD3) with our conventional dual–colour protocol. No significant differences were found between percentage of CD3+ lymphocytic cells determined with three different antibody combinations. When the CD8/CD4/CD3 combination was used a systematic overestimation of CD3+ CD4+% cells was found. This turned out to be caused by the formation of ‘CD8–escapees’. These are clumps of CD8+ cells that fall outside the lymphocyte gating region, principally because of high side scatter. The problem can be overcome by rigorous vortexing to loosen aggregates. The lymphocyte gating principle used in this protocol (gating on a side scatter/CD45 dot plot) is readily applicable to other antibody combinations. This was demonstrated by measuring CD5+ B lymphocytes, a subset receiving increasing attention in the study of HIV–induced immune deviations. We conclude that our three–colour protocol for CD4+ T–lymphocyte determinations offers significant advantages to the conventional dual–colour method, and we suggest that when possible anti–CD45 be added to dual–colour combinations in order to improve lymphocyte gating.