bluuebunny/crossref_metadata_2025 huggingface dataset:
Mannan‐Binding Protein Forms Complexes with α‐2‐Macroglobulin. A Proposed Model for the Interaction
Authors: STORGAARD P. , NIELSEN E. HOLM , SKRIVER E. , ANDERSEN O. , SVEHAG S.‐E.
Published: July 2006

We report that α‐2‐macroglobulin (α2M) can form complexes with a high molecular weight porcine mannan‐binding protein (pMBP‐28). The α2M/pMBP‐28 complexes were isolated by PEG‐precipitation and affinity chromatography on mannan‐Sepharose, protein A‐Sepharose and anti‐IgM Sepharose. The occurrence of α2M/pMBP‐28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti‐α2M affinity column and chelating Sepharose loaded with Zn2+. The eluates from these affinity columns showed α2M subunits (94 and 180 kDa) and pMBP subunits (28 kDa) in SDS‐PAGE, which reacted with antibodies against α2M and pMBP‐28, respectively, in Western blotting. Furthermore, the α2M/pMBP‐28 complexes were demonstrated by electron microscopy, Fractionation of pMBP‐containing D‐mannose eluate from mannan‐Sepharose on Superose 6 showed two protein peaks which reacted with anti‐C1 s antibodies in ELISA, one of about 650–800 kDa, which in addition contained pMBP‐28 and anti‐α2M reactive material, the other with an Mr of 100–150 kDa. The latter peak revealed rhomboid molecules (7 × 15 nm) in the electron microscope and a 67 kDa band in SDS‐PAGE under reducing conditions. This band was also seen in eluates from the anti‐α2M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP‐28 with α2M.
Normal Human Cord Blood B Cells can Produce High Affinity IgG Antibodies to dsDNA that are Recognized by Cord Blood‐Derived Anti‐Idiotypic Antibodies
Authors: WARRINGTON R. J. , WONG S.‐K. , RAMDAHIN S. , RUTHERFORD W. J.
Published: July 2006

It is possible to identify, in Epstein‐Barr virus‐transformed normal human cord blood B cell populations, cells present at a low frequency that produce IgG antibodies specific for dsDNA. By cloning out these B cells as immortalized monoclonal cell lines, it could be shown that the antibodies were the products of CD5 positive B cells. Two monoclonal anti‐dsDNA antibodies were derived from cell lines T52 and A7 and these were further characterized as anionic (pI ∼ 6.4) IgG4k antibodies that bound with affinities of 7.18 × 109 1/mol and 3.28 × 109 1/mol, respectively, to dsDNA but did not bind to ssDNA. These affinities were similar to those of polyclonal IgG anti‐dsDNA antibodies from lupus patients, which ranged from 1 × 109‐8.9 × 1010 1/mol. Both T52 and A7 monoclonal anti‐dsDNA antibodies were recognized by cord blood‐derived IgM antibodies. These IgM antibodies were not rheumatoid factors but bound to the F(ab')2 of A7 and T52 while failing to recognize T50, which is an autologous IgG4k monoclonal antibody without specificity for dsDNA. A cloned B cell line A24 generated from the same cord blood sample as A7 produced an IgM monoclonal antibody that bound to the heavy chains of T52 and A7, but not T50 on Western blot and inhibited the binding of these antibodies to dsDNA. A7 and T52 competitively inhibited each other in their binding to the anti‐idiotype A24, and A24 inhibited the binding to dsDNA of some polyclonal IgG anti‐dsDNA antibodies purified from sera of lupus patients. The level of inhibition of binding of these antibodies to dsDNA was directly proportional to the levels of expression of the idiotype recognized by A24 on these antibodies. The normal human cord blood, therefore, may contain cells that form an idiotype/anti‐idiotype network in which the idiotype is expressed on IgG antibodies with specificity for dsDNA and the anti‐idiotype is an IgM antibody that binds to a heavy chain idiotope in such a way as to interfere with its interaction with dsDNA. The presence of a similar idiotype on some polyclonal anti‐dsDNA antibodies in lupus that are similarly inhibitable by the cord blood‐derived anti‐idiotype raises the possibility that this network may persist in later life and perhaps become dysfunctional in systemic lupus erythematosus.
Modulation of Antigen‐Antibody Complexations by Immunoglobulins
Authors: ANDERSSON K. , HANSSON U.‐B. , BJORKLUND J. , ALKNER U.
Published: July 2006

In this investigation, the modulating effects of non‐immune human IgG and rheumatoid factors (RFs) on antigen‐antibody complexations were studied. Non‐immune human IgG, as well as RF, were found to inhibit the binding of antigen to specific antibodies of both human and rabbit origin. In addition, human immunoglobulins were also able to modify the composition of preformed antigen‐antibody complexes. The effects were detected by immunological methods in two different antigen‐antibody systems (human serum albumin‐rabbit anti‐HSA and tetanus toxoid‐human anti‐TT). Changes in biological activities could be followed by employing enzymes (glucose‐6‐phosphate dehydrogenase and human placental alkaline phosphatase) as antigens. The outcome of the effects was found to be dependent on the ratio of antigen to antibody, the antigen‐binding properties of the antibody and its origin, and on the properties of the immunoglobulins added. The observed changes could not be explained only by the presence of specific antibodies in the immunoglobulin preparations. The ability of immunoglobulins to modulate antigen‐antibody complexations may provide a rationale for the large amounts of non‐specific immunoglobulins in the circulation by preventing premature precipitation and promoting the elimination of antigenic molecules.
Binding to Erythrocyte Complement Receptor Type 1 of BSA/ Anti‐BSA Complexes Opsonized by C4A3 or C4B1 in the Presence of Serum
Authors: KLINT C. , TRUEDSSON L. , STURFELT G.
Published: July 2006

An in vitro model with human serum and human 0 Rh‐negative erythrocytes was used for studies on preformed BSA/anti‐BSA complex binding to complement receptor type 1 (CR1). The serum used was first depleted of Clq, factor D and properdin, then of C3, C4 or both and finally reconstituted with the desired proteins (serum reagent). With varying C4 concentrations and 100% C3 present, binding curves obtained for the two C4 isotypes were similar. When the serum reagent was not reconsitituted with factor D and properdin there was no difference between the CR1 binding of normal serum and the partially reconsituted serum reagent, nor between the two C4 isotypes in this serum reagent. When C3 at 50% or 100% of normal concentrations was added to the serum reagent together with 100% C4A3 or C4B1, the C4B1‐opsonized complexes showed more binding than the C4A3‐opsonized complexes. At very low levels of C3 (<25%) the binding could not be distinguished from the background. The results suggest that the binding of complement opsonized antigen/antibody complexes to erythrocyte CR1 is mediated mainly by C3, originating from activation of the classical pathway, and that the difference in properties between C4A and C4B does not have a major influence.
V‐Region and Class Specific RT‐PCR Amplification of Human Immunoglobulin Heavy and Light Chain Genes from B‐Cell Lines
Authors: WATKINS B. A. , DAVIS A. E. , FIORENTINI S. , REITZ M. S.
Published: July 2006

We have designed and tested primers that amplify complete human kappa and lambda light chain genes, and human Fd fragments from gamma, mu and alpha heavy chain genes. These primers were tested for efficiency and specificity on monoclonal sources of human immunoglobulin RNA, obtained from human B‐cell lines of known immunoglobulin gene expression. Analysis of the sequences derived from these B‐cells confirms the specificity of the PCR primers and the extent of somatic mutation seen in different B‐cell malignancies supports existing concepts for differing aetiologies in the tumours concerned.
Suppression of Experimental Autoimmune Myasthenia Gravis After CD8 Depletion is Associated with Decreased IFN‐γ and IL‐4
Authors: ZHANG GUANG‐XIAN , MA CUN‐GEN , XIAO BAO‐GUO , BAKHIET M. , LJUNGDAHL Å. , OLSSON T. , LINK H.
Published: July 2006

CDS+ T cells can perform both Th1 ‐ and Th2‐like functions by producing cytokines such as interferonγ (IFN‐γ) and interleukin‐4 (IL‐4), as well as the immune response down‐regulating transforming growth factor‐β (TGF‐β), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8+ T cells results in the suppression of EAMG accompanied by the down‐regulation of AChR‐specific B cell responses and AChR‐reactive IFN‐γ secreting Th1‐like cells. To identify the involvement of IFN‐γ, IL‐4 and TGF‐β in the development of EAMG after CD8+ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8+ T cells resulted in decreased levels of IFN‐7 and IL‐4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF‐β mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8+ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).
Usage of β1 Integrin Ligands by B Cells is Developmentally Regulated in Avian Bursa
Authors: PALOJOKI E. , JALKANEN S. , TOIVANEN P.
Published: July 2006

Chicken B cell development takes place in a separate organ, the bursa of Fabricius, which provides the blood‐borne stem cells, a microenvironment specialized for B cell maturation. Therefore, chicken can be used as a model to study specifically the molecules and interactions which control the development of the B cell compartment. In this work, we studied expression, localization and function of β1 integrins on maturing B cells and bursal stroma. The expression of β1 integrins on B cells increases during the embryonic development and β1 integrin‐positive cells can be found both in the medulla and the cortex throughout the bursal development. The binding assays show that the attachment of B cells to stroma is mediated by β1 integrins. Binding to 10‐day‐old embryonic stroma is fibronectin‐independent, whereas fibronectin‐mediated binding takes place in an increasing manner during further embryonic maturation. After hatching fibronectin appears to be the main binding site for B cells. However, the epitope of β1 integrin which takes part in the adhesion of B cells to stroma and to fibronectin is not crucial for the homing of cells into the bursa in a cell transfer model. Our results indicate that the interaction of β1 integrins with their ligands is developmentally regulated in the bursa and suggest that B cell maturation may be partially controlled by this interaction.
The Neuropeptide Substance P does not Influence the Migration of B, T, CD8+ and CD4+ (‘Naive’ and ‘Memory’) Lymphocytes from Blood to Lymph in the Normal Rat
Authors: HEERWAGEN C. , PABST R. , WESTERMANN J.
Published: July 2006

Thoracic duct lymphocytes (TDL) continuously patrol through the body, facilitating immune responses at most sites. The neuropeptide Substance P might regulate immune responses by influencing the migration of TDL. Therefore, it was investigated whether Substance P affects the migration of thoracic duct B, T, CDS+ and CD4+ (‘naive’ and ‘memory’) lymphocytes from blood to lymph in vivo. Labelled TDL were either incubated with Substance P and then injected into normal rats, or incubated without Substance P and then injected into rats continuously receiving Substance P intravenously. The numbers of labeled B, T, CDS+ and CD4+ (‘naive’ and ‘memory’) lymphocytes were determined in blood and thoracic duct lymph for 1 and 5 days, respectively. Neither the in vitro incubation with Substance P nor its in vivo application influenced the disappearance of any lymphocyte subset from the blood or its reappearance in the lymph. In addition, continuous intravenous application of the Substance P antagonist CP 96.345 did not alter the volume or the lymphocyte number of the efferent lymph. The present study indicates that the nervous system does not influence immune responses via Substance P by altering the migration pattern of B, T, CD8+ and CD4+ (‘naive’ and ‘memory’) lymphocytes.
Secretion of MPB64 Antigen by a Recominant Clone of Mycobacterium Smegmatis: Characterization and Application for the Diagnosis of Tuberculosis
Authors: TASAKA H. , SHIGETO E. , MATSUO K. , YAMAGUCHI R. , HAGA S. , YAMAZAKI A. , YAMAZAKI T. , NAGAI S. , NAKAMURA R. M.
Published: July 2006

MPB64, a specific antigen to Mycobaeterium tuberculosis complex (TB complex), was produced and secreted by a clone of M. smegmatis‐MPB64 where the structural gene of MPB64 was inserted using a new mycobacteria, E. coli shuttle plasmid pNIS vector. Antibodies against the recombinant MPB64 (rMPB64) were used for the reverse particle latex agglutination (RPLA) test to detect the MPB64 antigen rapidly. RPLA tests were applied to the stock cultures and the clinical isolates of mycobacteria to identify TB complex. RPLA with anti‐MPB64 antibody‐coated latex beads completely distinguished TB complex from other mycobacteria. Thus, it is suggested that RPLA with anti‐MPB64 antibody would be a new, easy and inexpensive method for the rapid diagnosis of tuberculosis.
Nitric Oxide Synthase Pathway May Mediate Human Natural Killer Cell Cytotoxicity
Authors: XIAO L. , ENEROTH P. H. E. , QURESHI G. A.
Published: July 2006

The present study provides evidence that the human natural killer (NK) cell effector mechanism causing target cytolysis has a requirement for L‐arginine. In a deficient medium (DM) containing only salts, buffer system and glucose, NK cell‐mediated cytotoxicity was found to decrease by 70% as compared to that obtained in a complete medium (CM). However, adding L‐arginine to such DM could restore the activity of NK cells to the normal level. Many other components of CM, such as serum, glutamine and vitamins did not improve NK cell‐mediated killing in DM. When all amino acids except L‐arginine were added to DM only a partial recovery of NK cell functional cytolysis was seen. L‐arginine enhanced the NK cell activity in a dose‐dependent manner. Additionally, the inhibitor of both inducible and constitutive nitric oxide synthase, N‐monomethyl‐L‐arginine (L‐NMMA) inhibited NK cytolytic activity in DM supplemented with L‐arginine indicating participation of nitric oxide (NO). The results also show that the stimulatory effect of L‐arginine on human NK cell‐mediated cytotoxicity was accompanied by an increase in NO formation as determined by accumulation of nitrite and citrulline. L‐NMMA gave a dose‐dependent reduction in NO generation as well. The nitrite and citrulline production dose‐dependenlly correlated with not only the concentration of L‐arginine in the cultivation medium, but also the enhanced NK cell‐mediated cytolysis. Taken together, these findings could define a L‐arginine/NO‐linked effector mechanism in human NK cells. Nitrite and citrulline were not formed when NK cell‐mediated target cell killing took place in a L‐arginine‐free DM supplemented with additives. Thus, it appears as if human NK cells may cause target cell killing via both NO‐dependent and ‐independent processes.
Down‐Regulation of CD59 (Protectin) Expression on Human Colorectal Adenocarcinoma Cell Lines by Levamisole
Authors: BJØRGE L. , MATRE R.
Published: July 2006

The vulnerability of tumour cells to complement‐mediated immune attack is regulated by membrane associated molecules. Recently, we have shown that the expression of the membrane attack complex inhibitor CD59 is enhanced on colonic adenocarcinoma cells compared to normal colonic epithelial cells. CD59 was shown, in the same study, to protect the tumour cells from complement‐mediated lysis. Levamisole (LMS), used in conjunction with 5‐fluorouracil as adjuvant therapy, reduces the incidence of colon cancer relapse following surgical resection. This led to our investigation of the effect of LMS on CD59 expression and function on the human colorectal cell lines HT29 and Caco‐2. When cultured in the presence of 10 μM LMS, the cells reduced their expression of CD59 in a time‐dependent manner. LMS treated HT29 ceils were more sensitive to lysis by complement than control cells, and the reduction in CD59 expression was shown to be partly responsible for this. A reduction in CD59 expression will augment complement‐mediated immune surveillance and may contribute to LMSs anti‐tumour activity in vivo.
Characterization of Monoclonal Antibodies to the αIIbβ3 Integrin on Bovine Platelets
Authors: NTHALE J. M. , SYFRIG J. , PEARSON T. W. , NAESSENS J.
Published: July 2006

A set of monoclonal antibodies (MoAbs) to leucocyte antigens is an essential tool to identify different cell types and functional membrane molecules involved in immune responses. Since no MoAbs existed to bovine integrins, except against the β2 subfamily, we generated MoAbs to β3 integrin after the immunization of mice with bovine platelets. Two MoAbs, IL‐A164 (IgG2a) and IL‐A166 (IgG1), were selected that reacted specifically with bovine platelets and detected the same membrane molecule. The antigen was a heterodimer of two polypeptide chains of 122 kDa and 95 kDa as resolved by SDS‐PAGE under reducing conditions. Although the Mr of the smaller subunit is identical to that of β2 integrin, pre‐absorption with an antibody to β2 (or CD18) did not remove the bovine antigen. Comparing the molecular masses of the two subunits in reduced and non‐reduced forms showed a pattern that was similar to that of human GPIIb/IIIa (also called αIIbβ3 or CD41a). Reduction of the bovine molecule increased the apparent Mr of the light chain from 76 kDa to 95 kDa, while the heavy subunit changed from 136 kDa to 122 kDa. As with human GPIIb, the decrease in Mr of the α‐subunit is probably a result of a small disulphide‐linked polypeptide, although no additional evidence for this was detected for the bovine integrin. Sequencing of the N‐terminal amino acids of both bovine polypeptides showed identity of the bovine integrin with human GPIIb/IIIa.
Short‐Term Administration of Selected Anti‐T‐Cell Receptor Vβ Chain Specific MoAb Reduces Sialadenitis in MRL/lpr Mice
Authors: SKARSTEIN K. , HOLMDAHL R. , JOHANNESSEN A. C. , GOLDSCHMIDT T. , JONSSON R.
Published: July 2006

Sialadenitis develops spontaneously in MRL/Mp mice bearing a lymphoproliferative gene, lpr (MRL/ Mp‐lpr/lpr). Based on recent observations of an oligoclonal expansion of T‐cell receptor (TCR) expressing Vβ chain families (Vβ4, Vβ8.1, 2, Vβ10b) in salivary glands of these mice we have initiated selective antibody therapy. Treatment with monoclonal antibodies (MoAb) specific for T cells expressing a mixture of TCR Vβ4, Vβ8.1, 2 and Vβ10b was applied to MRL/lpr mice before and after the spontaneous development of Sialadenitis. The in vivo treatment with Vβ4, Vβ8.1, 2 and Vβ10b MoAb did not prevent the development of Sialadenitis. However, in animals with established Sialadenitis, treatment with the MoAb significantly decreased the inflammation compared with the control groups, Immuno‐histochemical staining of cell phenotypes demonstrated a change in the ratio of CD4/CD8 in the animals with established Sialadenitis. Altogether, these findings illustrate that it is possible to modulate Sialadenitis and infiltrate cell phenotypes in vivo in MRL/lpr mice with specific anti‐TCR Vβ MoAb treatment.
A Single Major Transcript Encodes the Membrane‐Bound Form of Rat Immunoglobulin E
Authors: AVESKOGH M. , HELLMAN L.
Published: July 2006

The primary structure of the membrane‐bound form of rat immunoglobulin E was determined by PCR amplification and nucleotide sequence analysis of its mRNA. The sequence was found to correspond to the previously identified membrane exons of the rat ɛ chain gene. The donor splice site in the C4 exon was mapped to a position 35 nt upstream of the stop codon for the secreted form of rat IgE. Therefore, the membrane‐bound IgE lacks the 12 C‐terminal amino acids present in the secreted form of the protein. Recently, five novel ɛ chain transcripts were isolated from human IgE producing B‐cells or B‐cell lines. Four of these transcripts encode proteins which differ in their C‐terminal ends from the classical membrane or secreted forms of human IgE. To investigate if these transcripts were likely to represent functional mRNAs, their evolutionary conservation was studied by screening a rat IgE producing B‐cell line for the expression of similar transcripts. By PCR amplification and cloning of transcripts, containing both the C3 and the M2 exons, approximately 10, 000 independent cDNA clones were obtained. These clones were screened with probes directed against regions specifie for each of the five novel human epsilon chain mRNAs. However, no evidence was found for the presence of transcripts with a similar structure, indicating that no specific function associated with these transcripts and their corresponding proteins has been conserved between human and rat. The lack of additional M2‐containing transcripts in the rat suggest that the novel human IgE transcripts are byproducts of differential splicing and that they most likely encode proteins with no evolutionarily important function.
Lack of Functional Similarity Between Complement Factor H and Anticardiolipin Cofactor, β2‐Glycoprotein I (Apolipoprotein H)
Authors: PUURUNEN M. , JOKIRANTA S. , VAARALA O. , MERI S.
Published: July 2006

Beta 2‐glycoprotein I (β2‐GPI) is a 50 kDa protein in human plasma composed of five repeating complement control protein modules thereby closely resembling complement factor H which has 20 such units. Both β2‐GPI and factor H (150 kDa) have binding sites for negatively charged polyions. β2‐GPI has been shown to act as a cofactor for antiphospholipid antibodies upon their binding to anionic phospholipids. In factor H the polyanion recognition site participates in the discrimination between alternative pathway activating and non‐activating surfaces. In light of the structural similarity between β2‐GPI and factor H we have examined whether β2‐GPI has a role in the alternative complement pathway recognition process. Both activators (zymosan) and non‐activators (sheep erythrocytes) of the alternative complement pathway were coated with C3b. Radiolabelled factor H was observed to recognize C3b on both surfaces, whereas β2‐GPI bound to neither. In competition experiments β2‐GPI could not prevent the association of 125I‐H with either non‐activator or activator bound C3b. Conversely, factor H could not replace β2‐GPI as a cofactor for antiphospholipid antibodies upon their binding to anionic phospholipids. It is concluded that β2‐GPI and factor H, despite similarities in structure, exhibit distinct, non‐overlapping functions.
Both Immunization with Protein and Recombinant Vaccinia Virus Can Stimulate CTL Specific for the E7 Protein of Human Papilloma Virus 16 in H‐2d Mice
Authors: ZHU X. , TOMMASINO M. , VOUSDEN K. , SADOVNIKAVA E. , RAPPUOLI R. , CRAWFORD L. , KAST M. , MELIEF C. J. M. , BEVERLEY P. C. L. , STAUSS H. J.
Published: July 2006

The transforming protein E7 of human papilloma virus type 16 can stimulate cytotoxic T lymphocytes (CTL) which can protect experimental animals against growth of E7 expressing tumour cells. In this study we compared CTL responses in mice immunized with either E7 protein in MF59 adjuvant or with recombinant vaccinia virus expressing E7 (Vac‐E7). We have chosen H‐2d mice because no E7‐specific CTL responses have been described in this MHC haplotype. Immunization of these mice with Vac‐E7 generated CTL which lysed target cells infected with Vac‐E7 or transfected with the E7 gene. CTL from mice immunized with E7 protein in MF59 adjuvant showed specificity for the same target cells. Antibody blocking experiments revealed that both immunization with Vac‐E7 and E7 protein stimulated CD8+ effector CTL. The find specificity of CTL induced by the two immunization protocols was similar. A major CTL epitope was mapped to the carboxyl terminal amino acids 48–98 of the E7 protein. Peptide isolation from E7 expressing cells followed by HPLC separation indicated that CTL induced by immunization with protein and Vac‐E7 recognized the same HPLC purified peptide fractions. Together, the study suggests that vaccines based on protein can activate CTL with similar fine specificity to CTL induced by vaccines based on recombinant vaccinia virus.
The Importance of C4A Null Genes in Norwegian Patients with Systemic Lupus Erythematosus
Authors: SKARSVÅG S.
Published: July 2006

C4A null genes were determined by RFLP (Taq I) and SSO‐probing on PCR‐amplified C4‐DNA in 51 Scandinavian patients with systemic lupus erythematosus (SLE) and 124 controls. Associations of the alleles DRB1*0301, DQA1*0501, DQBl*0201 had previously been found in this SLE group, as well as increased frequency of HLA‐DRB1 and ‐DQ homozygosity. The frequency of the allele C4A*Q0 was increased among the patients (RR = 2.3, P = 0.0172). The SSO‐probing revealed additional cases of C4A*Q0 homozygotes among the controls, leading to diverging RR values for C4A*Q0 homozygotes depending on the technique used. The RFLP method gave an RR of 9.7 (P = 0.0028), while the SSO‐probing resulted in an RR of 4.8 (P = 0.0153), demonstrating that unprecise characterization of C4A*Q0 in a relatively small material has great effect on the calculated RR. Multiple 2 × 2 tests were performed in an attempt to detect the strongest association of the alleles DRB1*0301, DQA1*0501 and C4A*Q0 (in linkage disequilibrium). These comparisons showed a trend towards stronger association for DQA1*0501 and DRB1*0301 than for C4A*Q0, and no interaction between the HLA alleles and the allele C4A*Q0. This may suggest that HLA class II molecules themselves and/or an unknown susceptibility gene located near the DQA1 and DRB1 loci are involved in the pathogenesis of SLE.
Specific Interferon‐Gamma Producing CD4+ and CD8+ T Cells After Autologous EBV‐B Stimulation: The Necessity of Restimulation
Authors: VOOIJS W. C. , NOOTEBOS M. G. , WERNERT H. , DB GAST G. C.
Published: July 2006

The response of T cells to produce interferon‐γ, to proliferate and to become cytotoxic after specific stimulation with low dose (2%) autologous EBV‐B cells was investigated in 15 EBV seropositive and five seronegative patients. A significantly higher number of interferon‐γ producing cells (56 ± 24 per 105 T cells) were found in a spot ELISA in EBV positive than in EBV negative patients (7 ± 2 spots, P<0.01) and it was only found with restimulation after 5–12 days of primary culture. No correlation was found between the extent of interferon‐γ production, cytotoxicity or profileration. Specificity of EBV‐induced interferon‐γ production was demonstrated by comparison of the response to allogeneic EBV‐B cells or IL‐2 in the reslimulation phase. The response was stronger in CDS+ T cells than in CD4+ T cells and could be blocked in the restimulation phase with HLA class I and class II antiserum, respectively.
Differential Patterns of T‐Cell Receptor BV‐Specific Activation of T Cells by gp120 from Different HIV Strains
Authors: AKOLKAR P. N. , GULWANI‐AKOLKAR B. , SILVER J.
Published: July 2006

Studies by several groups have suggested that HIV infection in vivo results in a BV‐specific alteration of the TCR repertoire and that this might play a role in the pathogenesis of AIDS. Our earlier studies demonstrated tbat both a crude extract of HIV451 as well as purified gp160 from HIV451 could specifically activate, in vitro, T cells expressing a common set of TCRBV segments (TCRBV3, 12, 14, 15, and sometimes BV17 and 20) in individuals of disparate HLA type. Furthermore, purified gp120 from HIV451 was shown to have a similar ability to activate T cells, although with a slightly diiferent TCRBV‐specific pattern. In order to determine whether gp120 from other HIV strains could similarly activate T cells in a TCRBV‐specific pattern, PBMC from HIV seronegative individuals of disparate HLA type were stimulated with gp120 from three strains of HIV (451, IIIB, and MN). The authors found that gp120 from all three strains activate T cells bearing TCRBV2 and BV3 in nearly every individual. T cells expressing other BV segments are also activated, but this is more variable and appears to be unique to each individual. Furthermore, gp120451 and gp120 from HIVIIIB and HIVMNdiffer in their ability to activate T cells expressing these other TCRBV segments. These observations suggest that variation in the structure of gp120 and in the genetic and/or environmental background of the individual play an important role in determining which TCRBV segments are‘triggered' by gp120. Furthermore, these observations may have important implications for the rate of disease progression in HIV‐infected individuals.
The Antibody Response to Secondary Immunization with Pneumococcal Polysaccharides in Mice
Authors: AABERGE I. S. , LØVIK M.
Published: July 2006

The benefit of re‐immunization with pneumococcal polysaccharide vaccine is an important question in clinical practice. In an experimental model, BALB/c and CBA/J mice were re‐immunized s. c. with a 23‐valent pneumococcal polysaccharide vaccine at various time intervals after a first immunization with the same vaccine. The antibody response after the secondary immunization showed similar kinetics as after primary immunization, and was mainly an IgM antibody response. Re‐immunization at 28 days or earlier induced a decrease in the serum antibody levels to the vaccine. Reimmunization at 120 days or later induced higher antibody levels than after the first immunization. Significant increases in antibody levels to serotypes 1, 4, 7F and 19F out of six serotypes tested were observed. In CBA/J mice, but not in BALB/c mice, the dose used for primary immunization appeared to influence the magnitude of the antibody response to secondary immunization. Our results indicate that the time interval between primary and secondary immunization is an important determinant with regard to the magnitude of the antibody response to re‐immunization with pneumococcal polysaccharide vaccine.
Adhesion of Plasmodium Falciparwn‐lnfected Erythroeytes to Human Cells and Seeretion of Cytokines (IL‐1‐β, IL‐1RA, IL‐6, IL‐8, IL‐10, TGFβ, TNFα, G‐CSF, GM‐CSF)
Authors: WAHLGREN M. , ABRAMS J. S. , FERNANDEZ V. , BEJARANO M. T. , AZUMA M. , TORII M. , AIKAWA M. , HOWARD R. J.
Published: July 2006

The scientific interest in tbe pbysical interaction of Plasmodium falciparum‐infected erytbrocytes with host cells stems from the suggestion ibat excessive binding in the microvasculature leads to severe malaria. Tbe authors studied, therefore, two parasites for their ability to adbere to normal human cells and to induce cytokine production, one parasite lacking a binding capacity (DD2) and one which adhered to CD36+ transfected CHO cells (MCAMP). The MCAMP parasites readily bound to platelets and erytbrocytes and to monocytes, polymorphonuclear granulocytes and EBV‐transformed B cells as seen by ligbt and electron microscopy. Platelets were frequently attached in large numbers to the infected erythrocyte surface and groups of infected erytbrocytes were sometimes held together by several platelets. Nine out of 17 cytokines tested were found to be secreted into the culture supernatants after 35 h of co‐eultures containing monocytes or unfractionated peripheral blood mononuclear cells (PBMC) and parasites (IL‐lRA, IL‐6, IL‐8, IL‐10, TGFβ, TNFα, G‐CSF, IL‐1‐β, and GM‐CSF). Three additional cytokines were also present in low levels (<200pg/ml, IL‐2, IL‐4, IFNγ) in tbe culture supernatants after incubation of the cells for 4 days. TNFa, IL‐RA, and IL‐8 were secreted from polymorphonuclear granulocytes, LGLs and T cells. Platelets and, to a lesser degree, monocytes and T cells secreted large amounts of TGFβ (10–30 ng/ml). Cytokines may participate in tbe patbogenesis but also the suppression of immune responses seen during acute malarial infections.
Immunoparasitological Studies of Trypanosoma Cruzi Low Virulence Clones from Panama: Humoral Immune Responses and Antigenic Cross‐Reactions with Trypanosoma Rangeli in Experimentally Infected Mice
Authors: SALDAÑA A. , SOUSA O. E. , ÖRN A.
Published: July 2006

The kinetics of humoral immune responses were investigated in mice experimentally infected with five clones of Trypanosoma cruzi isolated from different sources in Panama. Sera were collected at different timepoints post‐infection. ELISA and IHA tests were used to detect antibodies against T. cruzi epimastigote antigens. The levels of T. cruzi specific antibodies increased during the course of infection; at day 90 post‐infection the range was between 1:5120 and 1: 10240. A high correlation was evident between ELISA and IHA results. Western blots revealed that these antibodies recognized polypeptides of 81, 76 and 71 KDa during the first weeks and 81, 76, 71, 50, 40, 28 and 12 KDa after 30–50 days. Only minor differences in antigen recognition patterns were demonstrated, suggesting that the major antigens may be represented in all clones. T. rangeli antigens were also recognized by T. cruzi seropositive sera. However, an ELISA test using antigens isolated from a genomic expression library of T. cruzi revealed that a hyperimmune rabbit serum against T. rangeli was unable to recognize the repeat sequence of SAPA (Shed Acute Phase Antigen) peptides but did recognize a number of other T. cruzi synthetic peptide antigens. The importance of these findings, in the context of Chagas' disease, is discussed.
Preferential Positive Selection of T Lymphocytes which Express Two Different TCRα Chains, an Endogenous and a Transgenic
Authors: MUNTHE L. A. , SOLLIEN A. , DEMBIC Z. , BOGEN B.
Published: July 2006

A hallmark of positive selection in T‐cell receptor (TCR)‐transgenic mice is a strong skewing towards the CD4+ or the CD8+ subset, depending on the class II or I restriction of the TCR, respectively. However, previous experiments in TCR transgenic mice specific for an Ig light chain (λ2315)/I‐Ed class II molecule did not fit into this scheme because the authors observed an anomalous skewing towards CD8. In this paper, the authors show that endogenous TCRα chains are expressed on > 90% of CD4+ and CDS+ cells in this particular transgenic strain, even on a selecting H‐2d haplotype. Endogenous TCRQ chains are first detected when double‐positive thymocytes down‐regulate either CD4 or CD8. Endogenous Vα seems to influence generation of T‐cell subsets because CD4+ and CD8+ cells express different frequencies of endogenous Vα2 and Vα8. In the absence of endogenous TCRα chains in recombination‐deficient TCR‐transgenic severe combined immunodeficiency (SCID) mice, a strong skewing towards CD4+ T cells is seen, but such mice are severely T‐cell deficient. As an explanation for these results, the authors suggest that the transgenic TCR has a too low affinity for efficient positive selection, therefore, TCRa gene rearrangements proceed. Endogenous TCRa paired with transgenic TCRβ could bind to class I or class II molecules, enhance positive selection and thereby production of CD4+ or CD8+ cells. Most of the ‘mismatched’ CD8+ cells are λ2315‐specific and I‐Ed class II restricted, and may function as idiotype‐specific suppressors of B cells. These results may help explain the origin of dual TCRα T cells. Furthermore, the authors suggest that T cells ‘mismatched’ for co‐receptor/TCR MHC‐specificity may be enriched among dual TCRa T cells.
In Vitro Stimulation with Glutamic Acid Decarboxylase (GAD65) Leads to an Oligoclonal Response of Peripheral T‐Cells in an IDDM Patient
Authors: WEISS U. , MANFRAS B. J. , TERJUNG D. , EIERMANN T. , WÖLPL A. , LÖLIGER C. , KÜHNL P. , BOEHM B. O.
Published: July 2006

The enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in insulin‐dependent diabetes mellitus (IDDM). To study T‐cell reactivity towards GAD, peripheral blood leucocytes from seven patients with IDDM and five control subjects were stimulated in vitro with recombinant GAD. All diabetics studied were heterozygous for diabetes‐associated HLA alleles, i. e. HLA‐DRB1*03, *04‐DQBl*0302, *0201. A single IDDM subject (no. GAD65.05) revealed a strong response against GAD65. After stimulation, his T‐cell receptor β (TCRBV) usage was found to be oligoclonal. The sequence analysis of the putative peptide binding region of the T‐cell receptor (CDR3 region) of 37 GAD‐reactive T‐cell clones revealed no common CDR3 motif. The stimulation of GAD‐reactive T‐cells could be inhibited with anti‐class II monoclonal antibodies, indicating a class II restricted T‐cell response. In addition, GAD65‐responsive T‐cells revealed a Thl cytokine response pattern. The author's data suggest that GAD‐reactive T‐cells of Thl phenotype can be obtained after in vitro stimulation of peripheral blood leucocytes from an HLA‐DRBl*03/*04 heterozygous IDDM patient. The lack of a common CDR3 motif suggests the absence of an immunodominant T‐cell epitope in that patient, or may indicate receptor repertoire spreading of peripheral T‐lymphocytes.
C3 is Activated in Hereditary Angioedema, and C1/C1‐Inhibitor Complexes Rise During Physical Stress in Untreated Patients
Authors: NIELSEN E. W. , JOHANSEN H. T. , GAUDESEN Ø. , ØSTERUD B. , OLSEN J. O. , HØGÅSEN K. , HACK C. E. , MOLLNES T. E.
Published: July 2006

Seven patients witb hereditary angioedema (HAE) were studied to understand further how physical exercise may induce attacks. The most pronounced differences between patients and controls, however, were independent of the controlled bicycle run (mean values in patients/controls); C4(g/L): 0.12/0.28 (P= 0.0122); C4bc (AU/ml): 137.0/18.0 (P = 0.0002); C4d (mg/mL): 5.03/2.35 (P = 0.0004); C3bc (AU/ml): 8.4/6.3 (P= 0.0049); C3a (AU/ml): 11.1/5.6 (P= 0.0102). The ratio C4bc to C4 was 1141 versus 64. Consequently, a substantial part of the low amount of C4 left in HAE patients consists of activation products, and the authors show for the first time that a mild but significant activation of C3 occurs in HAE. The two HAE patients treated with danazol had values of Cl‐INH function and antigen, C4, and C2 in‐between those of normal and untreated patients, and lower levels of split products from C4 and high molecular weight kininogen than untreated patients. As a result of the exercise, fibrinolysis increased significantly in both patients and controls, while C1/Cl‐INH complexes rose significantly only in the five HAE patients without treatment when compared to the seven controls (P= 0.0089). This study thus suggests that complement activation is enhanced in untreated HAE patients following physical stress.
Lipopolysaccharide Effectively Up‐Regulates B7‐1 (CD80) Expression and Costimulatory Function of Human Monocytes
Authors: SCHMITTEL A. , SCHEIBENBOGEN C. , KEILHOLZ U.
Published: July 2006

The influence of lipopolysaccharide (LPS) and various cytokines on the expression of the costimulatory molecule B7‐1 and intercellular adhesion molecule‐1 (ICAM‐1), lymphocyte function associated antigen‐3 (LFA‐3) and human histocompatibility leucocyte antigen‐DR (HLA‐DR) on human monocytes and their effect on the costimulatory function was investigated. Freshly isolated human monocytes constitutively express ICAM‐1, LFA‐3 and HLA‐DR, but no B7‐1. B7‐1 expression was up‐regulated by LPS and, to a lesser extent, by interferon‐γ (IFN‐γ). The other stimuli tested, including IFN‐α, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor‐α (TNF‐α) and GM‐CSF + TNF‐α, did not influence expression of B7‐1 on monocytes. ICAM‐1 and HLA‐DR were up‐regulated by IFN‐γ and LPS; LFA‐3 expression was not influenced. LPS also effectively enhanced costimulatory function of monocytes as determined in the tetanustoxoid (TT) assay. Blocking of B7 by CTLA‐4Ig inhibited the LPS‐induced enhancement of costimulatory function almost completely. Our results indicate that the LPS‐mediated up‐regulation of the costimulatory function of human monocytes is mediated by B7. This mechanism may be important for host defence against Gram‐negative bacteria.
The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy†
Authors: Samanani Nailish , Alcantara Joenel , Bourgault Richard , Zulak Katherine G. , Facchini Peter J.
Published: July 2006

The benzylisoquinoline alkaloids of opium poppy, including the narcotic analgesics morphine and codeine, accumulate in the multinucleate cytoplasm of specialized laticifers that accompany vascular tissues throughout the plant. In mature opium poppy plants, immunofluorescence labeling using specific antibodies showed that four alkaloid biosynthetic enzymes, (S)‐norcoclaurine 6‐O‐methyltransferase (6OMT), (S)‐coclaurine N‐methyltransferase (CNMT), (S)‐3′‐hydroxy‐N‐methylcoclaurine‐4′‐O‐methyltransferase (4′OMT) and salutaridinol‐7‐O‐acetyltransferase (SAT) were restricted to sieve elements of the phloem adjacent or proximal to laticifers. The identity of sieve elements was confirmed by (i) the specific immunogold labeling of the characteristic cytoplasm of this cell type, (ii) the co‐localization of a sieve element‐specific H+‐ATPase with all biosynthetic enzymes and (iii) the strict association of sieve plates with immunofluorescent cells. The localization of laticifers was demonstrated antibodies specific to major latex protein (MLP), which is characteristic of this cell type. In situ hybridization using antisense RNA probes for 6OMT, CNMT, 4′OMT and SAT showed that the corresponding gene transcripts were found in the companion cell paired with each sieve element. Seven benzylisoquinoline alkaloid biosynthetic enzymes, (S)‐N‐methylcoclaurine 3′‐hydroxylase (CYP80B1), berberine bridge enzyme, codeinone reductase, 6OMT, CNMT, 4′OMT and SAT were localized by immunofluorescence labeling to the sieve elements in the root and hypocotyl of opium poppy seedlings. The abundance of these enzymes increased rapidly between 1 and 3 days after seed germination. The localization of seven biosynthetic enzymes to the sieve elements provides strong support for the unique, cell type‐specific biosynthesis of benzylisoquinoline alkaloids in the opium poppy.
UPF1 is required for nonsense‐mediated mRNA decay (NMD) and RNAi in Arabidopsis
Authors: Arciga‐Reyes Luis , Wootton Lucie , Kieffer Martin , Davies Brendan
Published: July 2006

Aberrant mRNAs containing premature termination codons (PTCs) have the potential to be translated into truncated proteins, which could act to the detriment of the organism by interfering with normal cellular processes. Eukaryotes have mechanisms of mRNA quality control that identify PTC‐containing transcripts and target them for destruction, a process known as nonsense‐mediated mRNA decay (NMD). Surprising differences have been reported in the mechanisms of NMD between different organisms. UPF1 and UPF3 are structurally unrelated proteins, which function in the NMD pathway in yeast, mammals, Drosophila and Caenorhabditis elegans. Here we show that NMD in plants requires UPF1, as mRNAs containing PTCs become stabilized in upf1‐5 mutants. However, in contrast to NMD in mammals, UPF1‐dependent NMD is capable of targeting both spliced and unspliced PTC‐containing mRNAs. An allelic series of upf1 mutants exhibits a range of unexpected vegetative and floral abnormalities, including jagged leaves, late flowering, fused flowers and seedling lethality. We also show that mutants in UPF3 share these abnormalities. As both UPF1 and UPF3 are required for NMD, the similar phenotypes of the upf1 and upf3 mutants suggest that NMD regulates a common set of genes required for plant development and survival. Finally, gene silencing by an inverted repeat transgene is impaired in upf1‐5 mutants, indicating a connection between UPF1 and RNA interference in plants.
A C2H2‐type zinc finger protein, SGR5, is involved in early events of gravitropism in Arabidopsis inflorescence stems
Authors: Morita Miyo T. , Sakaguchi Keitaro , Kiyose Shin‐ichiro , Taira Kensuke , Kato Takehide , Nakamura Moritaka , Tasaka Masao
Published: July 2006

Plants can sense the direction of gravity and change the growth orientation of their organs. To elucidate the molecular mechanisms of gravity perception and the signal transduction of gravitropism, we have characterized a number of shoot gravitropism (sgr) mutants of Arabidopsis. The sgr5‐1 mutant shows reduced gravitropism in the inflorescence stem but its root and hypocotyl have normal gravitropism. SGR5 encodes a zinc finger protein with a coiled‐coil motif. The SGR5–GFP fusion protein is localized in the nucleus of Arabidopsis protoplasts, suggesting that SGR5 may act as a transcription factor. Analysis of GUS expression under the control of the SGR5 promoter revealed that SGR5 is mainly expressed in the endodermis, the gravity‐sensing tissue in inflorescence stems. Furthermore, the observation that endodermis‐specific expression of SGR5 using the SCR promoter in the sgr5‐1 mutant restores shoot gravitropism indicates that it could function in the gravity‐sensing endodermal cell layer. In contrast to other sgr mutants reported previously, almost all amyloplasts in the endodermal cells of the sgr5‐1 mutant sedimented in the direction of gravity. Taken together, our results suggest that SGR5 may be involved in an early event in shoot gravitropism such as gravity perception and/or a signaling process subsequent to amyloplast sedimentation as a putative transcription factor in gravity‐perceptive cells.
A trial of phenome analysis using 4000 Ds‐insertional mutants in gene‐coding regions of Arabidopsis
Authors: Kuromori Takashi , Wada Takuji , Kamiya Asako , Yuguchi Masahiro , Yokouchi Takuro , Imura Yuko , Takabe Hiroko , Sakurai Tetsuya , Akiyama Kenji , Hirayama Takashi , Okada Kiyotaka , Shinozaki Kazuo
Published: July 2006

Mutant lines covering all Arabidopsis genes allow us to pursue systematic functional genomics. A comprehensive phenotype description, called a phenome, is highly sought after in the profiling of ‐omics data. We selected 4000 transposon‐insertional lines with transposon insertions in their gene‐coding regions, and systematically observed the visible phenotype of each line. For the first 3 weeks after germination, plants were grown on agar plates and the juvenile phenotypes were recorded. Then the plants were transferred to soil and their phenotypes were recorded at each growth stage. About 140 lines showed clear and reproducible visible phenotypes, including novel phenotypic mutants as well as previously reported ones. All descriptions of the mutants showing visible phenotypes were classified into eight primary categories (seedling, leaves, flowering and growth, stems, branching, flowers, siliques and seed yield) and 43 secondary categories of morphological phenotypes. Phenotypic images have been entered into a searchable database (http://rarge.gsc.riken.jp/phenome/). One example investigated through the use of plural alleles was a mutant of a novel gene related to glycerolipid biosynthesis, with a unique visible phenotype of sepal opening. Our results suggest that we can find more novel visible phenotypes and their corresponding genes, and that phenotypic mutants of gene knockouts are not exhausted yet. This study provides basic data on large‐scale phenotyping of gene knockout lines in plants, and will contribute to the completion of an international effort to develop a phenome database of all the functional genes in Arabidopsis.