bluuebunny/crossref_metadata_2025 huggingface dataset:
The Binding of Synovial Tissue‐Derived Human Monoclonal Immunoglobulin M Rheumatoid Factor to Immunoglobulin G Preparations of Differing Galactose Content
Authors: SOLTYS A. J. , HAY F. C. , BOND A. , AXFORD J. S. , JONES M. G. , RANDEN I. , THOMPSON K. M. , NATVIG J. B.
Published: July 2006

There are several sites on IgG Fc that have been reported to be the epitopes for binding rheumatoid factors (RF). It is now established that there are alterations in the oligosaccharides on IgG from patients with rheumatoid arthritis and it has been suggested that these changes may enhance immune complex and cryoglobulin formation.

We have used a series of IgG preparations differing in their content of oligosaccharide chains lacking galactose from 18 to 86% to determine whether changes in sugar content affect the binding of rheumatoid factor. Five of 16 monoclonal rheumatoid factors prepared from synovial tissue, from patients with juvenile or adult rheumatoid arthritis, bound better to IgG which was deficient in galactose. Six of the 16 rheumatoid factors from the same patients bound independently of the galactose content. Four of the 16 rheumatoid factors could not be absolutely grouped in this manner but seemed to demonstrate a preference for agalactosyl IgG. One rheumatoid factor bound better to fully galactosylated IgG. There was an association between enhanced binding to galactosc‐deficient IgG and monoreactivity and a very strong association between the functional affinity of the rheumatoid factors and the dependent binding.
A Rad LV‐Induced γδ T Cell Lymphoma Displaying an Antitumoral Cytotoxicity
Authors: MARTIN‐SIMONET M.‐T. , MARINI V. , RICCIARDI‐CASTAGNOLI P. , ASTIER‐GIN T. , GREIMERS R. , BONIVER J. , SCHAAF‐LAFONTAINE N.
Published: July 2006

We described previously the induction by RadLV infection of a lymphoma (NS8) expressing a cytolytic activity against an MCA‐induced fibrosarcoma. We report here that the cytolytic activity of these immortalized CD3+, CD8+ T cells is non‐MHC‐restricted. We then determined the structure and expression of the TCR chains expressed by these cells. Only partial rearrangement of the β chain associated to an abnormally short transcript was detected in NS8 cells, whereas the γ chain is rearranged and normally transcribed. On the opposite, rearrangement and expression of these genes were found in the other RadLV‐induced lymphomas analysed. Moreover, γ TCR proteins were detected on the cell surface of NS8 cells only, whereas the αβ complex, presents on the other T cell lines, was not expressed by NS8 cells.

The ability of NS8 cells or of cells obtained from activated lymph nodes (harvested from mice grafted with the T2 sarcoma used to induce the NS8 line) to lyse the T2 sarcoma cell line was analysed. With both types of lymphocytes, the cytotoxicity was partially inhibited by a preincubation of the effector cells with anti‐γ antibodies.

These results demonstrate that γδ lymphocytes can mediate anti‐tumour cytotoxicity and NS8 lymphoma line may be representative of the TCR γ CD8+ T cell subpopulation expressing non MHC‐restricted cytotoxicity and displaying antitumoral activity.
Cloning, Sequencing and Expression in Escherichia coli of the Gene for α Antigen from Mycobacterium scrofulaceum*
Authors: TAKANO M. , OHARA N. , MIZUNO A. , YAMADA T.
Published: July 2006

The gene for the extracellular a antigen of Mycobacterium scrofulaceum (S‐α) was cloned by using the α antigen gene fragments of Mycobacterium bovis BCG as probes. The complete nucleotide sequence was determined. The gene was expressed in Escherichia coli. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein. The deduced amino acid sequences were highly homologous to the α antigen of the species of other mycobacteria. Interestingly, the a antigens of MAIS complex (Mycobacterium avium—Mycobacterium intracellulare—M. scrofulaceum) were closely homologous even at the C‐terminal regions which were variable among those of M. bovis BCG, Mycobacterium leprae and Mycobacterium kansasii.
Comparative Analysis of the Antibody Response to the HTLV‐I gag and env Proteins in HTLV‐I Asymptomatic Carriers and HAM/TSP Patients: an Isotype and Subclass Analysis
Authors: DEKABAN G. A. , KING E. E. , ARP J. , PALKER T. J. , RICE G. P. A.
Published: July 2006

We have compared the immunoglobulin isotype and IgG subclass and the titre of neutralizing antibody responses to the human T celt lymphotropic virus type I (HTLV‐I) between a group of asymptomatic HTLV‐I infected individuals and a group with the neurological disease HTLV‐I associated myelopathy/tropical spastic paraparesis (HAM/TSP). A western blot titration assay and an envelope peptide ELISA were used to determine the presence and titre of isotype and IgG subclass responses to the gag p19 and p24 proteins and to the envelope protein. Significant increases were observed in the number of individuals seropositive for a particular isotype and IgG subclass in the HAM/TSP group versus the asymptomatic group particularly for IgM and IgE and to a lesser extent, IgA. The predominant IgG subclasses to the HTLV‐I p19, p24 and envelope proteins were IgG1 and IgG3. This finding was also observed in the titres of the antibody responses to these HTLV‐I proteins. The HAM/TSP group also exhibited significantly higher neutralizing antibody titres than the asymptomatic group. This evidence suggests that some form of chronic immune stimulation might be involved in the immunopathogenesis of HAM/TSP. In addition, by following the Western blot titre to the IgM and IgE isotypes in particular, it may be possible to identify asymptomatic individuals progressing to HAM/TSP.
Bacteriostatic Activity of BCG/PPD‐Activated Macrophages Against Mycobacterium fortuitum Does Not Involve Reactive Nitrogen or Oxygen Intermediates
Authors: NIBBERING P. H. , YOSHIDA S.‐I. , VAN DEN BARSELAAR M. T. , VAN FURTH R.
Published: July 2006

Mycobacteria preferentially reside in resident macrophages whereas activated macrophages are presumed to eliminate the bacteria effectively, The aim of the present study was to determine the antibacterial activities of resident and activated murine peritoneal macrophages against Mycobacterium fortuitum and the intracellular mechanisms involved. After phagocytosis M. fortuitum could not be killed by either BCG/PPD‐activated and IFN‐γ‐activated macrophages and resident macrophages. The mycobacteria did not multiply in BCG/PPD‐activated macrophages and the rate of proliferation of M. fortuitum in IFN‐γ‐activated macrophages was only slightly inhibited compared to that in resident macrophages. Experiments with selective inhibitors of the production of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) demonstrated that these factors are not essential for the mycobacteriostatic activity of BCG/PPD‐activated macrophages. After phagocytosis of M. fortuitum, BCG/PPD‐activated and IFN‐γ‐activated macrophages produced substantial amounts of both RNI and ROI. No correlation was found between the levels of these intermediates and the proliferation of M. fortuitum in the macrophages. In conclusion, BCG/PPD‐activated macrophages are bacteriostatic, but not bacteriocidal for M. fortuitum and the former does not involve reactive nitrogen and oxygen intermediates.
Familial Macroglobulinaemia: Hyperactive B‐Cells but Normal Natural Killer Function
Authors: ÖGSMUNDSDÓTTIR H. M. , JÓHANNESSON G. M. , SVEINSDÓTTIR S. , EINARSDÓTTIR S. , HEGEMAN A. , JENSSON Ó.
Published: July 2006

An Icelandic family with two cases of benign monoclonal gammopathy and one case each of Waldenström's macroglobulinaemia, histiocytic lymphoma and multiple myeloma was first described in 1978. Nine family members had then shown raised values for se‐IgM. Of these one has since died and another was not available for testing. In four of the remaining seven se‐IgM had returned to normal; the three subjects who still showed raised se‐IgM included the case of multiple myeloma diagnosed in 1985. Baseline production of IgM, IgG and Ig in vitro was normal in the 35 family members studied compared with 13 healthy control subjects, but the mean production of all immunoglobulin classes in response to minimal stimulation with PWM (1 μg/ml) was significantly increased (P <0.05). Ten family members showed markedly increased production of all three immunoglobulin classes (> 3 × SD above mean for controls). Raised production of IgM never occurred alone, indicating intact class switching. One family member showed extremely high values: IgA: 5.15 μg/ml, IgG: 16.3 μg/ml, IgM: 24.8 μg/ml (means for controls: 0.066, 0.123, 0.185 respectively). These 10 family members were of both sexes, ranged in age from 16 to 84 years and were clustered mainly in three distinct groups within the pedigree suggesting heredity. Proliferative responses to PWM were not significantly increased. Serum levels of interleukin‐4 were tested in the patient with multiple myeloma and the family member with highest Ig production and found to be normal. We found no evidence for depressed NK function. Thus, in this family with a tendency for macroglobulinaemia and B cell derived malignancies B cell hyperreactivity was detectable by in vitro testing in several asymptomatic family members, of both sexes and a11 ages. No evidence was obtained for defects in regulatory mechanisms.
Interactions of Opsonized Immune Complexes with Whole Blood Cells: Binding to Erythrocytes Restricts Complex Uptake by Leucocyte Populations
Authors: NIELSEN C. H. , SVEHAG S.‐E. , MARQUART H. V. , LESLIE R. G. Q.
Published: July 2006

The binding of opsonized, fluorescein‐labelled bovine serum albumin (BSA)/rabbit anti‐BSA complexes (IC) to washed human whole blood cells and isolated leucocytes in the presence of autologous serum was investigated by flow cytometry. In the presence of erylhrocytes (E), the IC‐binding to granulocytes (PMN), monocytes and lymphocytes was inhibited by up to 46%, 61% and 48%, respectively, depending on the incubation time and the IC‐concentration tested. The E‐mediated inhibition of the binding to PMN was found to correlate with the average numbers of CR1 per E during the initial 15 min of incubation. Thereafter, the difference between IC binding to PMN in absence and presence of E, decreased in accordance with decreasing binding to E. IC‐uptake by PMN induced a drop in side‐scatter characteristics, attributable to degranulation, which could be prevented by the presence of E. In contrast to the findings for PMN, the difference between IC‐binding to monocytes in the absence and presence of E increased progressively over the 90 min observation period, suggesting that different mechanisms are involved in the late‐phase IC uptake by monocytes and PMN. Lymphocytes were heterogeneous with respect to IC binding, the main contributors being B cells. E initially inhibited and then later enhanced the IC binding to lymphocytes, suggesting that E promote B cell uptake of C3d, g‐covered IC via CR2. Our findings, that E can restrict the IC uptake by circulating leucocytes, and that an IC‐induced degranulation of PMN may be prevented by E, indicate that E may act as a high capacity buffer limiting inappropriate activation of phagocytes by circulating IC
Fcγ‐Receptor Activity of Placental Annexin II
Authors: KRISTOFFERSEN E. K. , ULVESTAD E. , BJØRGE L. , AARLI Å. , MATRE R.
Published: July 2006

We have previously produced a MoAb, B1D6, against a plaeental FcR. The antigen isolated using F(ab')2‐fragments of B1D6 exhibits Fc‐binding properties with low affinity for IgG. The antigen is a single‐chained glycoprotein with a molecular weight of approximately 37 kDa and a pi of about 7.0‐8.5. Amino acid sequences from enzymatic digests of the antigen indicated that it is annexin II. Immunoreactivity using anti‐annexin antisera and purified placental annexin II have further established the specificity of BID6 to annexin II. The B1D6 epitope appears to be intramembraneous and intracellular on placental syncytiotrophoblasts, monoeytes and other cells investigated.
The Importance of G1m and 2 Allotypes for the IgG2 Antibody Levels and Avidity Against Pneumococcal Polysaccharide Type 1 Within Mono‐ and Dizygotic Twin‐Pairs
Authors: KONRADSEN H. B. , OXELIUS V.‐A. , HAHN‐ZORIC M. , HANSON L. Å.
Published: July 2006

Eighty‐two mono‐or dizygotic Caucasian twins vaccinated with a 23‐valent pneumococcal vaccine, who had previously had their IgG2 antibody levels to pneumococcus type 1 determined before and after vaccination, were included in this study. Their IgG2 antibody levels were related to their G1m and G2m allotypes/phenotypes and their Gm amounts.

Eight different Gm phenotypes were found and characteristically IgG2 antibody levels were related to them. G2m (n) homozygotic twins had significantly higher IgG2 levels than heterozygotic twins who had significantly higher levels than G2m (‐n) homozygotic twins (P <0.05). The G1m allotype, on the other hand was without influence on the IgG2 levels and so were the Gm amounts among G2m (n) heterozygotic twins. The IgG2 antibody avidities were not related to Gm allotypes but significantly correlated to IgG2 levels (P = 0.05). Finally, a highly significant intra‐pair correlation was found for avidity in the monozygotic twins supporting a genetic regulation of avidity (P <0.002).

These results may explain our earlier findings that IgG2 antibody levels after pneumococcal vaccination are significantly more closely correlated within mono‐compared to dizygotic twins.
The Intersubunit Region of the Influenza Virus Haemagglutinin is Recognized by Antibodies During Infection
Authors: NAGY Z. , RAJNAVÖLGYI É. , HOLLÓSI M. , TÓTH G. K. , VÁRADI G. , PENKE B. , TÓTH I. , HORVÁTH A. , GERGELY J. , KURUCZ I.
Published: July 2006

The influenza virus haemagglutinin has an important role in the infectious cycle of the virus and carries multiple B and T cell epitopes. It is synthesized as a single polypeptide chain but viral infectivity depends on its post‐translational enzymatic cleavage. The cleavage site of a trypsin‐like enzyme responsible for this modification is found in the most conserved intersubunit region of the molecule. In this study the role of this region in antibody recognition was investigated. Synthetic peptides comprising the intact and cleaved forms of the intersubunit segment were used to examine the specificity of virus‐ or peptide‐ induced antibodies. The immune response elicited by viral infection resulted in the appearance of antibodies capable of neutralizing the virus without interfering with its binding to the receptor. A monoclonal antibody (MoAb) of such functional properties was shown to recognize the intact intersubunit region both in the uncleaved haemagglutinin molecule and in a 25‐mer synthetic peptide comprising the intact intersubunit region. Specificity and functional studies revealed the conformation dependent recognition of the C‐terminal segment of the haemagglutinin 1 subunit by this MoAb. The binding of the antibody was shown to inhibit the trypsin‐mediated cleavage of the haemagglutinin molecule and the membrane fusion event. The enzymatic cleavage of the haemagglutinin was demonstrated to abolish antibody recognition of the infective virus suggesting an escape mechanism mediated by the functional destruction of this highly conserved region. The synthetic peptide corresponding to the intact intersubunit region is characterized by an ordered structure and is able to elicit an antibody response in BALB/c mice while its subfragments are nonimmunogenic. Furthermore, this peptide elicited a protective immune response demonstrated by in vivo experiments.
Serum Amyloid A Protein in Mink During Endotoxin Induced Inflammation and Amyloidogenesis
Authors: BRUUN C. FOYN , RYGG M. , NORDSTOGA K. , SLETTEN K. , MARHAUG G.
Published: July 2006

Two‐dimensional electrophoresis was used to study SAA and AA proteins in mink during lipopoly‐saccharide‐induced inflammation and amyloidogenesis. Three isotypes, SAA pI 6.8 and SAA pI 6.5 (both SAA1‐like), and SAA pI 6.0 (SAA1‐ and SAA2‐like), were identifled in serum after both single and multiple LPS injections. Total SAA serum levels were highest in the early phase of induction, followed by a decrease ranging from 1 to 50% of the peak value during the rest of the experiment. The variation in the total SAA levels correlated with the total SAA mRNA levels. Low total SAA levels were seen both in non‐amyloidotic and amyloidotic animals, and a general decrease of all isotypes was demonstrated. In hepatic amyloid fibrils, several AA isotypes, with amino acid sequence homologous exclusively to that of SAA2, were found. In the corresponding splenic material, fragments of histones H2A and H2B constituted most of the low molecular mass proteins, and no protein AA was detected. In spite of low serum levels and a non‐specific isotype removal, the results confirm that SAA2 is amyloidogenie in mink.
A Human Rheumatoid Factor C304 Shares VH and VL Gene Usage with Antibodies Specific for Ubiquitous Human Viral Pathogens
Authors: RIOUX J. D. , RAUCH J. , SILVESTRI L. , NEWKIRK M. M.
Published: July 2006

Analysis of the variable region getie sequetices of a human hybridoma rheumatoid factor (RF), derived from a patient with rheumatoid arthritis (RA), revealed the expression of genes from the VβI and VH3 families. Specifically, the C304 RF had rearranged the DPL8/Humlv1042 and VH26 germline VL and VH genes, respectively. This gene usage has been observed in the rearrangement of human anti‐viral antibodies specific for the herpes group of viruses. This overlap between the autoimmune and anti‐viral antibody gene repertoires suggests a possible structural relationship between the immune response directed against ubiquitous pathogens and the induction of RF production.
The Role of CD4+ T Cells in Cell‐Mediated Immunity to LCM V: Studies in MHC Class I and Class II Deficient Mice
Authors: CHRISTENSEN J. P. , MARKER O. , THOMSEN A. R.
Published: July 2006

Parameters of the virus‐specific T‐cell response were analysed in order to dissect the contribution of CD4+ and CDS+ T cells to cell‐mediated immunity to lymphocytic choriomeningitis virus. In MHC class II deficient mice, initial T‐cell responsiveness was not impaired, but virus clearance was delayed, and virus‐specific Td activity declined more rapidly. Furthermore, class I restricted Tc memory appeared to be impaired in these mice. To directly evaluate the role of CD4+ cells in virus clearance and T‐cell mediated inflammation, MHC class I deficient mice were also studied. No virus‐specific delayed‐type hypersensitivity reaction was detected following infection of the footpad, and only a few mice died from intracerebral challenge. However analysis of markers of T‐cell activation as well as direct evaluation of CSF infiammation unveiled a low degree of T‐cell activation and a chronic cellular exudate. This low‐grade response was associated with some degree of virus control as organ titres were lower in these animals than in matched T‐cell deficient nu/nu mice or class I deficient mice treated with anti‐CD4 monoclonal antibody. This confirms that CD4+ cells are not needed to induce a virus‐specific CD8+ T‐cell response, but our findings strongly suggest that CD4+ T cells are critical for maintaining full antiviral immunity. Furthermore, CD4+ T cells per se have a low potential for mediating virus‐specific infiammation that is associated with a low degree of virus control.
Production and Characterization of Monoclonal Antibodies Against the Major Cysteine Proteinase of Trypanosoma cruzi
Authors: GONZALEZ G. , ÖRN A. , CAZZULO J. J. , GRÖNVIK K.‐O.
Published: July 2006

In the present study we describe the production and characterization of a panel of monoclonal antibodies (MoAbs) directed against cruzipain (Crz), the major cysteine proteinase from Trypanosoma cruzi. The five MoAbs, BD6, BF2, CG2, CH8, and DC10 were analysed with respect to affinity and specificity. None of the MoAbs cross‐reacted with papain, which has regions of high homology with Crz. Treatment of the antigen with periodate did not affect the binding of the MoAbs. suggesting that they bind to the polypeptide moiety of Crz. CH8 recognized a continuous epitope located at the C‐terminal extension of the proteinase that appeared to be highly immunogenic. Although the rest of the MoAbs recognized epitopes located in the catalytic domain, the enzymatic activity of Crz was not impaired by the binding of the MoAbs. Characterization of the antibody‐binding sites revealed the presence of at least four separate epitopes.
Differential Role of Protein Kinase C in Cytokine Induced Lymphocyte‐Endothelium Interaction In Vitro
Authors: EISSNER G. , KOLCH W. , MISCHAK H. , BORNKAMM G. W. , HOLLER E.
Published: July 2006

In the present study we investigated the influence of the PKC‐inhibitor GF109203X on cytokine‐ and endotoxin‐induced expression of intercellular adhesion molecule 1 (ICAM‐1) and on the adhesion of lymphocytes to cytokine‐activated endothelial cells. We found that tumour necrosis factor alpha (TNF‐α)‐ and lipopolysaccharide (LPS)‐induced ICAM‐I expression on a human endothelium‐derived cell line (EA. hy926) were unaffected by the PKC‐inhibitor and thus appeared to be independent of PKC activation. In contrast, GF109203X significantly reduced ICAM‐1 expression induced by interferon‐γ (IFN‐γ) and interleukin‐1 (IL‐1). The functional relevance of these findings was evaluated in an adhesion assay using human umbilical vein endothelial cells (HUVEC) and peripheral blood mono‐nuclear cells (PBMC). In fact, the IFN‐γ‐ and IL‐1‐induced adhesion of PBMC to cytokine treated HUVEC could be down‐regulated by the PKC‐inhibitor, whereas TNF‐α and LPS‐mediated adhesion was not affected. Additionally, the IL‐1‐driven ICAM‐1 expression on HUVEC as well as the IL‐I induced adhesion of PBMC to HUVEC was found to be TNF‐dependent, as both effects could be inhibited by an anti‐TNF‐α monoclonal antibody (MoAb) (MAK195). Based on these data on differential regulation of cytokine‐induced lymphocyte‐indothelium interactions our study supports the use of PKC‐inhibitors as additive modulators in cytokine related pathophysiological conditions.
Interleukin‐10 Inhibits Antimicrobial Activity Against Leishmania major in Murine Macrophages
Authors: VIETH M. , WILL A. , SCHRÖPPEL K. , RÖLLINGHOFF M. , GESSNER A.
Published: July 2006

The stimulation of macrophages is of importance to the defense against intracellularly replicating microorganisms such as Leishmania. In this study the direct effect of recombinant interleukin‐10 (IL‐10) on the leishmanicidal effector functions of murine peritoneal or bone marrow derived macrophuges was investigated. IL‐10 almost completely inhibited the killing of intracellular leishmania at concentrations above 10ng/ml. This inhibitory effect was independent of the stimulus used as the activation of macrophages by IFN‐γ and IL‐7, recently shown to possess macrophage activating properties, were suppressed by IL‐10. Kinetic experiments revealed that IL‐10 must be present during the process of macrophage activation and that the leishmanicidal effector function of fully activated macrophages was not influenced. Furthermore, in the absence of exogenously added IL‐10, the addition of neutralizing antibodies against IL‐10 or IL‐10‐specific antisense phosphorothioate DNA‐oligonucleotide led to an enhanced killing of parasites after stimulation with either IFN‐7 or IL‐7. In accordance with this, IL‐10 mRNA was readily detectable in murine macrophages by PCR with reverse transcribed mRNA. These results indicate that IL‐10, which is endogenously produced by macrophages, acts as an autocrine deactivating factor supporting the survival of the parasite.
Analysis of the Capacity to Produce IL‐3 in Murine AIDS
Authors: NEUENSCHWANDER A. U. , MARKER O. , THOMSEN A. R. , Thomsen Allan Randrup
Published: July 2006

Adult C57BL/6 mice infected with LP‐BM5 murine leukaemia virus represent a model of murine AIDS (MAIDS). In this study we have analysed the capacity of CD4+ T cells from infected mice to produce IL‐3 following stimulation with ConA for 24–72 h. In contrast to the position with IL‐2, the production of which is markedly impaired during LP‐BM5 infection, similar levels of IL‐3 were measured in culture supernatants of splenocytes from infected and uninfected mice harvested at 24 h of stimulation. Forty eight and 72 h of ConA stimulation led to increasing levels of IL‐3 being measured in cultures from uninfected mice, whilst in cultures from infected animals, IL‐3 levels remained stagnant. Similar results were obtained 4, 8 and 13 weeks post‐infection. In view of the fact that parallel experiments revealed markedly impaired proliferative responses to ConA during MAIDS, we conclude that IL‐3 production is basically intact at the cellular level in T cells during MAIDS; but when in a situation requiring clonal expansion of the activated T cells, IL‐3 production will be inhibited owing to the impaired capacity for proliferation.
Rheumatoid‐Factor‐Reactive Sites on CH2 Established Analysis of Overlapping Peptides of Primary Sequence
Authors: WILLIAMS R. C. , MALONE C. C.
Published: July 2006

Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis (RA) isolated on IgG affinity columns were studied for their reactivity with overlapping 7‐mer peptides representative of solvent accessible regions of the CH2 domains of IgG using a pin‐ELISA assay. A panel of nine monoclonal RF from RA patients' B cells were studied in parallel. Four peptides SVFLFPP (239–245), KFNWYVD (274–280), NSTYRVVSV (297–305) and VLTVLHQNWL (305–314) reacted with most RF. Glycine substitution showed that tryptophanes at 277 and 313, tyrosine at 278, valines at 279 and 305, and leucine 314 represented important residues for RF reactivity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a restricted spectrum of reactivity for CH2 epitopes, which often were identical to those binding to polyclonal IgM RF. Combinations of synthetic 7‐mer peptides representing RF‐reactive CH2 or CH3 epitopes produced as much as 60–90% inhibition of RF binding to IgG when peptides were preincubated with RF in free solution before completion of the reaction of RF with IgG on the ELISA plate.
Kinetics of Somatic Mutation in Lymph Node Germinal Centres
Authors: KÄLLBERG E. , GRAY D. , LEANDERSON T.
Published: July 2006

Somatic mutation activity in immunoglobulin Vk genes during the response to the hapten 2‐phenyl‐5‐oxazolone was measured in lymph node B‐cell populations at various timepoints after footpad immunization. When the VkOxl genes rearranged to the Jk5 segment were amplified from genomic DNA using the polymerase chain reaction and sequenced, somatic mutations could be detected as early as day 4 after immunization. Somatic mutations were also detected after sequencing RNA from oxazolone‐specific hybridomas derived from lymph node cells at day 4 after immunization. These early mutations were found mostly in cells with a germinal centre phenotype. No indication of selection at the population level by apoptosis was detected until day 7 after immunization. These results suggest somatic mutations can be induced very early during the immune response in lymph node cells, prior to the peak of clonal expansion and selection with regard to antigen binding.
Correlations of Monocyte Phagocytic Receptor Expressions with Serum Immune Complex Level in Systemic Lupus Erythematosus
Authors: SZÜCS G. , KÁVAI M. , SURÁNYI P. , KISS E. , CSIPÖ I. , SZEGEDI G.
Published: July 2006

The expression of FcγRI, FcγRII, and FcγRIII (the IgG receptors CD64, CD32, CD16) as well as CR3 (the C3bi receptor, CD11b) on monocytes in the blood of patients with systemic lupus erythematosus (SLE) was investigated. The relationship between the receptor expression and the serum immune complex (IC) concentration was analysed. The decrease in mean fluorescence intensity (FI) of the FcγRII of patients' monocytes stained by specific monoclonal antibodies (MoAb IV3) was very close to statistical significance (P= 0.052). The expression (FI) of CR3 (using MoAb OKM1) on monocytes of patients was also decreased, but not significantly. The detected decrease of FcγRII and CR3 was inversely correlated with the high circulating immune complex level in patients' sera. At the same time, FcγRI expression on SLE monocytes (using MoAb 32) was significantly elevated and this change was in parallel with the serum IC concentration.
Secondary IgE Responses In Vivo are Predominantly Generated Via γ1ɛ‐Double Positive B Cells
Authors: VAN OMMEN R. , VREDENDAAL A. E. C. M. , SAVELKOUL H. F. J.
Published: July 2006

We have recently developed a model in which mice were treated with 1L‐4 after primary immunization, resulting in elevated total serum IgG1 and IgE levels, but decreased antigen‐specific levels and memory formation for these isotypes. In this report, we describe that these effects of IL‐4 are mediated at the B cell and not the T‐cell level. Major changes occurred in the γ1ɛ‐double positive B‐cell population which is increased as a result of IL‐4 treatment. Moreover, it is shown that γ1ɛ‐double positive B cells can develop in vitro out of γ1‐positive primed B cells and that these double positive cells can differentiate into IgG1‐ and IgE‐secreting cells. The existence of γ1ɛ‐double positive memory B cells can explain the differences in cytokine dependence of TNP‐specific memory IgG1 and IgE responses found after adoptively transferring primed spleen cells into irradiated naive recipients. Whereas the IL‐4 independent TNP‐specific memory IgG1 responses could be blocked efficiently by neutralizing IL‐5 and IL‐6, TNP‐specific memory IgE responses were virtually not susceptible to such treatment. These IgE responses were also not susceptible to IFN‐γ, used in doses that could inhibit the primary IgE response. Inhibition of the TNP‐specific memory IgG1 response by neutralizing IL‐5 and IL‐6 is accompanied by a 10‐fold increase of the IL‐4 independent TNP‐specific IgE memory response. These data indicate that secondary IgE responses primarily result from B cells that are either switched to IgG1, or are double positive for IgG1 and IgE, thereby suggesting a minor role for ɛ‐single positive B cells in secondary IgE responses.
Activation of Protein Kinase C Induces De Novo Synthesis of the Soluble Interleukin‐6 Receptor in Human B Cells
Authors: KÖRHOLZ D. , NUSSBAUM P. , PAFFERATH B. , MAUZ‐KÖRHOLZ C. , HEMPEL L. , BURDACH S.
Published: July 2006

The mechanism of protein kinase C (PKC) induced release of the soluble interleukin‐6 receptor (sIL‐6R) from human B cells was investigated. Phorbot myristat acetat (PMA)‐induced activation of PKC significantly enhanced the release of sIL‐6R from the human B‐cell line SKW 6.4. The PMA effect was completely blocked by cycloheximide, whereas different inhibitors of proteases had no effect. In contrast to the effect on sIL‐6R release, FACS analysis did not reveal any effect of PMA on the expression of IL‐6R on the surface of SKW 6.4 cells. After 6h of stimulation with PMA, analysis of mRNA expression using a polymerase chain reaction‐(PCR)‐assisled mRNA amplification assay, showed increased expression of a spliced mRNA encoding for a soluble form of IL‐6R. Comparable to the results in SKW 6.4 cells, activation of purified human B cells with PMA induced a significant augmentation of sIL‐6R release which was also sensitive to cycloheximide. In conclusion, a novel mechanism of sIL‐6R release is reported involving de novo synthesis. Thus, sIL‐6R release from human B cells is completely different compared with that described in hepatocytes, which involved rapid, proteolytic cleavage of the membrane‐bound receptor but not de novo synthesis. The results of this study may help to understand the molecular control of sIL‐6R release from human B cells.
The Effects of Thymopentin on the Development of SLE‐Like Syndrome in the MRL/lpr‐lpr Mouse
Authors: NICOLETTI F. , ZACCONE P. , MAGRO G. , BARCELLTNI W. , CAVALLARO V. , BELLI G. , COCUZZA C. , Dl MARCO R. , MERONI P. L.
Published: July 2006

Thymopentin (TP‐5) is a synthetic pentapeptide that corresponds to the active 32–36 amino acid sequence of the thymic hormone thymopoietin, of which it retains all the immunomodulatory properties. In this study, we have evaluated the effects of long term prophylactic treatment with TP‐5 on the clinical, immunological and histological parameters of the SLE‐like syndrome that spontaneously occurs in MRL/lpr‐lpr (MRL‐lpr) mice. TP‐5., administered (s. c.) to these mice at the doses of 1, 10 and 100mg/kg. was given daily, five times a week, from the 9th to the 26th weeks of life. The prophylactic treatment with TP‐5 prolonged in a clear dose‐dependent fashion the lifespan of MRL‐lpr mice as compared with PBS‐treated control mice, and the effect reached statistical significance at the doses of 10 and 100mg/kg. In parallel ex vivo studies., this chnical effect was associated with multiple profound modifications of the immune system including: (i) the reduction of the spontaneous and Con A‐induced release of interleukin‐4 (IL‐4); (ii) the increased secretion of interferon‐γ (IFN‐γ) and IL‐6 upon polyclonal mitogenic stimulation, and (iii) the amelioration of the defective Con A‐induced lympho‐proliferative response. In contrast, although the drug diminished the severity of proteinuHa in MRL‐lpr mice, it neither reduced histological signs of lupus nephritis nor diminished the serum titres of antinative DNA and anti‐histone autoantibodies. These results indicate that TP‐5 displayed powerful immunomodulatory activities in a well known model of human SLE.
GD2 Ganglioside‐Specific Monoclonal Antibody Reacts with Murine Cytotoxic T Lymphocytes Reactive with FBL‐3N Erythroleukaemia
Authors: YOSHIMURA A. , TAKAMIYA K. , KATO I. , NAKAYAMA E. , SHIKU H. , FURUKAWA K.
Published: July 2006

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL‐3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4+ CD8−, CD4−CD8+, and CD4−CD8− phenotypes reacted with anti‐GD2 monoclonal antibody with various intensities. The reactivity with anti‐GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A‐treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.
Identical VHD and DJH Junctions in Monoclonal Antibodies Derived in Response to Dextran B512 Could be the Result of Developmental Selection
Authors: FERNANDEZ C.
Published: July 2006

We describe here the CDR3s of a collection of monoclonal antibodies (MoAb) with specificity for the carbohydrate dextran B512 produced in the mouse strain C57BL/6. In spite of the postulated mechanisms for variability in this region, a high proportion of these monoclonals displayed identical VHD (24/30) and DJH (21/30) junctions and 21 of them were identical in the whole CDR3. These 21 independently generated identical CDR3s could be ordered in eight groups indicating that not a particular CDR3, but instead the mechanism for generating identical junctions was preserved. Two of the CDR3s in this study were found to be identical to the CDR3 of the monoclonal B1‐8 produced in C57BL/6 in response to proteins bearing the hapten (4‐hydroxy‐3‐nitrophenyl)acetyl (NP). This and other parameters support the notion that the generation of identical junctions could be independent of antigenic selection. We also report here the association between JH usage and amino acid (aa) residues at the VHD and DJH junctions. Since these MoAb were generated in response to dextran B512, immunoglobulin conformation has to be compatible with antigen binding. Nevertheless, no aa residue of CDR3 could be directly related to antigen binding. We postulate therefore, that the observed selection of CDR3s could be directed to the production of variable regions with protein configuration most suitable with immunoglobulin folding and may occur prior to antigenic selection. Selection for junctional residues in relation to JH usage and the generation of identical CDR3s are probably different events. Possible genetic mechanisms operating for CDR3 construction and/or selection by cellular ligands are discussed.
Characterization of T‐Cell Receptor Vβ Repertoire in Ovarian Tumour‐Reacting CD3+ CD8+ CD4− CTL Lines
Authors: FISK B. , FLYTZANES C. N. , POLLACK M. S. , WHARTON J. T. , IOANNIDES C. G.
Published: July 2006

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL‐2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T‐cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3+ CD8+ CD4− cells from cultures of TIL and tumour‐associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3+ CD8+ CD4− lines of known HLA‐A, ‐B and ‐C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8+ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour‐reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour‐reactive CD3+ CD8+ CD4− CTL from different individuals of known HLA types.
Immunological Abnormalities in Patients with Chronic Fatigue Syndrome
Authors: TIRELLI U. , MAROTTA G. , IMPROTA S. , PINTO A.
Published: July 2006

Between January 1991 and January 1993, 265 patients who fulfilled the CDC criteria of the working case definition of Chronic Fatigue Syndrome (CFS) have been observed at our Institution and submitted for clinical and laboratory evaluation. One hundred and sixty‐three patients were females and 102 males, the median age was 35 years (range 4–55 years); all patients reported profound and prolonged fatigue, lasting for a median of 3 years (range 6 months–10 years), preceded or accompanied at appearance by fever in 185 cases, and neuropsychologic problems including inability to concentrate, difficulty in thinking, confusion, irritability, forgetfulness, and depression. The fatigue was so severe that it required 102 patients to stop their working activities for a period of time ranging from 3 months to 2 years (range 7 months). In 40 consecutive patients a comprehensive immunologic testing by single and two‐colour flow cytometry was performed and results compared with a group of 35 healthy, age‐ and sex‐matched controls. Whilst no significant differences were found in the absolute numbers of circulating total T cells (CD3+) and of total helper/inducer (CD4+) or suppressor/cytotoxic (CDS+) T cells, an evident reduction in CD3−/CD16+ and CD57+/CD56+ NK lymphocytes along with an expansion of the CD8+/CD56+ and CD16−/CD56+ NK subsets, were found in the CFS group. In addition, CD56+ NK cells from CFS subjects were found to express an increased amount of cell adhesion molecules (CD11b. CD1 1c, CD54) and activation antigens (CD38). Both the percentage and absolute numbers of CD4+ T cells bearing the CD45RA antigen appeared significantly reduced in CFS patients, and CD4+ T lymphocytes from CFS subjects displayed an increased expression of the intercellular adhesion molecule‐1 (ICAM− 1/CD54). Finally, the total numbers of circulating (CD19+) B lymphocytes, were significantly higher in CFS cases than in controls, and in 11 out of 30 CFS patients the increase in circulating B cells was sustained by the expansion of the CD5+/CD19+ subset of B lymphocytes. We conclude that CFS is a syndrome not previously described in Italy, with already known clinical characteristics and appears to be associated with several immunologic abnormalities, including those reported previously in cohort of patients from different countries. We also show for the first time that CD56+ NK cell subsets from CFS patients display an abnormally increased expression of cell adhesion molecules and activation markers.
Thl‐Like Human T‐Cell Clones Recognizing Leishmania gp63 Inhibit Leishmania major in Human Macrophages
Authors: KEMP M. , HEY A. S. , BENDTZEN K. , KHARAZMI A. , THEANDER T. G.
Published: July 2006

The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T‐cell clones recognizing this protein were generated from individuals who had previously had self‐healing cutaneous leishmaniasis. The T‐cell clones expressed CD4, and the alpha chain of the T‐cell antigen receptor. Gp63 reactive T‐cell clones activated by antigen or by immobilized anti‐CD3 antibody released relative large amounts of interferon‐gamma and no or little interleukin‐4, thereby resembling Th l cells. Autologous mononuclear cells and Epstein‐Barr virus‐transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells recognizing gp63 can take part in the host defence against L. major infections.
The Expression of Carbohydrate Antigens in Activated T Cells and in Autoimmune Diseases
Authors: FORTUNE F. , WALKER J. , LEFRANCOIS M. , LEHNER T.
Published: July 2006

Cell surface carbohydrate antigens have been implicated in cell differentiation and maturation and may play a role in immunoregulation. The expression of carbohydrates in peripheral blood lymphocytes (PBL) was studied by double immunofluorescence flow cytometry, using MoAbs CTI and CT2 but only a small proportion of cells bound these MoAbs. MoAbs CTI, CT2 and the lectin vicia villosa (VV) which share specificity for Gal NAc were then used to examine lymphocytes from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Behcet's disease (BD) and IgA nephropathy. A significant increase in MoAbs CTI CT2 and W binding CD4 or CD8 cells was found only with lymphocytes from patients with SLE. However, MoAbs CT or VV binding lymphocytes from healthy subjects were significantly up‐regulated by activation with a mitogen (PHA), cross‐linked anti‐CD3 MoAb or a common antigen (65kDa heat shock protein), suggesting that an increased proportion of T cells expressing these carbohydrates results from any of the three types of lymphocyte activating agents. Inhibitioti studies were then carried out to determine the relationship between the MoAbs CTI and CT2, VV and GalNAc. Indeed. VV binding to T cells was significantly inhibited by either MoAbs CTI or CT2, or GalNAc but not GlucNAc. suggesting that VV shares a common binding site with MoAb CT and that GalNAc may constitute one of the sugar receptors. Investigations of lymphocytes from adult peripheral blood in health and disease suggest that carbohydrate antigens may play a role in activation and immunoregulation.
Role of the Bowel Flora for Development of Immunity to hsp 65 and Arthritis in Three Experimental Models
Authors: BJORK J. , KLEINAU S. , MIDTVEDT T. , KLARESKOG L. , SMEDECÅRD G.
Published: July 2006

An infectious aetiology in rheumatoid arthritis (RA) has for long been suggested, although no conclusive evidence for this is at present available. Lately a large interest has been devoted to the involvement of heat shock proteins (hsps) in autoimmune disorders due to their conserved structure and immunogenic properties. Immunity to hsps has been observed both in human autoimmune conditions and in experimental models of autoimmune disease. We have studied the role of the bacterial flora and hsp immunity in the arthritic response in three experimental models of arthritis; type II collagen arthritis (CIA), adjuvant arthritis (AA) and oil induced arthritis (OIA); by using germ free and conventional DA rats.

A high incidence of severe arthritis developed in all the models evaluated irrespectively of whether the animals were in the conventional or germ free state. This confirms earlier results which show a minor effect of the bacterial flora in CIA and AA in high responder strains. These results also show that a severe OIA can develop in germ free animals. Despite the severe arthritic response induced, no serum antibody levels to hsp 65 could be detected in the germ free animals, which was in contrast to the conventional animals where a positive anti‐hsp 65 serum response was seen in 35–80% of the animals with CIA, AA or OIA.

These results show that development of a humoral response to hsp 65 in these models of arthritis is dependent on the presence of a bacterial flora. Further, the lack of humoral immunity in germ free animals despite a severe arthritic response indicates that humoral immunity to hsp 65 is not involved in development of disease in these three models of experimental arthritis.
Structural Specificity of MHC‐Unrestricted Recognition of HCMV‐Infected Target Cells by Human CD56+NK and LAK Cells
Authors: STEINMASSL M. , ANDERER F. A.
Published: July 2006

Structural specificity of binding and cytolysis of HCMV‐infected human foreskin fibroblasts (HFF) by human NK and LAK cells was studied in inhibition assays. A sample of 60%‐deacetylated α‐D mannose penta‐acetate was used as inhibitor that was previously shown to specifically inhibit binding and cytolysis of tumour target cells by human NK and LAK cells. We found now that cytolysis of HCM V‐infected HFF was inhibited in a dose‐dependent manner showing complete inhibition at concentrations above 640 nmoles/ml mannose acetate. This effect on cytolysis was based on inhibition of conjugate formation between virus‐infected cells and CD56+ NK and LAK cells. In the presence of mannose acetate (640 nmoles/ml) conjugate formation of virus‐infected cells was suppressed down to the level of uninfected cells. The latter showed residual conjugate formation on the basis of adhesive interactions with chemospecifity other than for mannose acetate, which were not capable of triggering cytolytic reactions. Coculturing of target cells with LAK cells appeared to induce expression of additional mannose acetate‐specific target sites yielding increases of conjugate formation and cytolysis.