bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective immunosuppression with anti‐interleukin 2 receptor‐targeted therapy: helper and suppressor cell activity in rat recipients of cardiac allografts
Authors: Kupiec‐Weglinski Jerzy W. , Padberg Winfried , Uhteg Lawrence C. , Ma Lan , Lord Roseanne H. , Araneda Dorian , Strom Terry B. , Diamantstein Tibor , Tilney Nicholas L.
Published: March 2007

(LEW × BN)F1 cardiac allografts are rejected within 8 days in unmodified LEW rats. ART18, a mouse anti‐rat IgG1 monoclonal antibody which binds specifically in vitro to the interleukin 2 receptor (IL2R) molecule expressed primarily on activated T cells, prolongs allograft survival in a dose‐dependent fashion to ca. 3 weeks (p < 0.001) after being administered for 10 days after transplantation. This effect was related to the specificity of the antibody for IL2R, as therapy with ART62 (a monoclonal antibody recognizing MHC class I antigen but not binding the rat IL2R) was ineffectual. Suppressor activity was detected in spleen cells of ART18‐treated grafted hosts: in vivo, splenic T suppressor/cytotoxic fraction adoptively transferred into normal LEW improved donor‐specific but not third‐party test graft survival (17 days, vs. 8 days, respectively, p < 0.001); in vitro, mixed lymphocyte reaction was profoundly but nonspecifically inhibited (< 5% of test mixed lymphocyte reaction, p < 0.001 as compared to acutely rejecting controls). In contrast, splenic T helper (Th) cells from ART18‐treated hosts were functionally depressed, as noted by their passive transfer into immunologically anergic B recipients of cardiac allografts (rejection in ca. 40 days, vs. ca. 13 days after transfer of Th from specifically sensitized rats). ART18 treatment also resulted in diminished elaboration of IL 2 as compared to normal (p < 0.005) or acutely rejecting hosts (p < 0.001); however, a remarkable increase in the production of IL 3 occurred (p < 0.001). These results demonstrate that IL 2R‐targeted therapy of immunocompetent graft recipients produces a selective immune defect in which donor‐specific T suppressor cells are spared, but Th cells attenuated or destroyed. Decreased elaboration of IL 2 concomitantly augments the release of IL 3, a lymphokine which might play a role in suppressor effect in vivo. In addition, IL 2R‐targeted therapy of the immunodeficient graft recipients abrogates the capacity of alloactivated T cells to re‐establish acute immune responsiveness.
Monoclonal antibodies to restricted and cross‐reactive idiotopes on monoclonal rheumatoid factors and their recognition of idiotope‐positive cells
Authors: Ono M. , Winearls C. G. , Amos N. , Grennan D. , Gharavi A. , Peters D. K. , Sissons J. G. P.
Published: March 2007

Four human monoclonal rheumatoid factors (MRF) were used to raise a panel of mouse monoclonal antibodies (mAb) which were selected in a solid‐phase radioimmunoassay for binding to MRF but not normal IgM. Three mAb, each raised against a different MRF, bound to the majority of MRF and also to most polyclonal RF. Four other mAb bound selectively to the MRF against which they were raised and to no other MRF, and rarely to any polyclonal RF. Competition studies using cold and radiolabeled mAb further indicated that these mAb recognize distinct and different epitopes on MRF. RF activity of MRF was inhibited by 3 of the 4 mAb binding to a single MRF and 2 of the 3 mAb binding to multiple RF. It was thus concluded that of this panel of mAb 3 recognized cross‐reactive idiotopes and the remainder demonstrated highly restricted idiotopes on MRF. These mAb identified MRF idiotopebearing cells in the peripheral blood of 3 of the MRF donors (and a further subject with type II essential cryoglobulinemia), with a frequency ranging from 0.3–10% of all mononuclear cells with the mAb to restricted idiotopes or 1.5–17% with mAb to cross‐reactive idiotopes. These anti‐idiotopic mAb should thus provide a highly specific means of identifying and monitoring MRF‐producing cells in vivo.
Hybridomas producing human monoclonal antibodies against varicella‐zoster virus
Authors: Sugano Toru , Matsumoto Yoh‐Ichi , Miyamoto Chikako , Masuho Yasuhiko
Published: March 2007

Hybridomas producing human monoclonal antibodies (mAb) against varicella‐zoster virus (VZV) were generated by fusing human splenic lymphocytes with mouse myeloma cells. Before cell fusion, lymphocytes were stimulated in vitro with viral antigens and pokeweed mitogen. This combination synergistically increased the generation of VZV‐specific hybridomas. Five established hybridomas have been stably producing mAb for at least 9 months. These mAb, designated V1, V2, V6, V8 and V9, were of the IgG1λ isotype. They bound to all 6 tested VZV strains but not to other herpes viruses, with the exception that V1 bound to herpes simplex virus (HSV) as well as VZV. Immunoprecipitation analysis showed that V1, V6 and V9 recognized glycoprotein gpII, whereas V2 and V8 recognized gpI. In addition, V1 reacted with the gB glycoprotein of HSV. All these mAb neutralized viral infectivity. The neutralizations by V2 and V8 were more effective and more complement dependent than those by V1, V6 and V9. Immunofluorescence tests revealed that all these mAb bound to the surface membrane of VZV‐infected cells. These results suggest that cell fusion between in vitro stimulated lymphocytes and mouse myeloma cells is a reliable method for the generation of hybridomas capable of stable production of human mAb. The human mAb thus developed may provide a new means of passive immunization of humans against VZV infection.
Sequences and repertoire of the human T cell receptor α and β chain variable region genes in thymocytes
Authors: Kimura Nobuhiro , Toyonaga Barry , Yoshikai Yasunobu , Du Ran‐Pan , Mak Tak W.
Published: March 2007

To compare and contrast the human T cell antigen receptor (TcR) α and β chain messages found in human thymocytes to those previously isolated from human peripheral blood T lymphocytes and other nonthymic sources, 13 TcR α and 13 TcR β cDNA were isolated from a human thymocyte library and the nucleotide sequences were determined. The data indicate that, as was found in the peripheral T lymphocytes, the majority of the TcR α and TcRβ chain thymocyte cDNA were derived from potentially functional messages. Although the thymocyte‐derived TcR cDNA do not contain any unique structural features when compared to TcR cDNA from mature T lymphocytes, 4 new Jα segments, 17 new V‐gene segments (9 Vα; 8 Vβ) and 7 additional V‐gene families (4 Vα and 3 Vβ) and sequences had been identified. The exon CβO, found in many murine thymocyte TcR β messages, was not found in over 75 human β chain messages. Based on these new data, a revised estimate of human TcR Vα, Jα and Vβ repertoires is calculated. The most significant change has been the increase in the estimated number of human TcR Vβ‐gene segments to a total of about 100 distributed among about 18 families. The Vα families are now revised upward to 16, with a total number of Vα segments of 50. The estimate of the Jα segments in humans remains between 50–100.
Degranulation of in vitro differentiated mast cells stimulated by two monoclonal IgE specificities
Authors: Rofolovitch Michal , Amira Milly , Ginsburg Haim
Published: March 2007

Confluent spread of mature nondividing mast cells is obtained after 1 month's growth of lymph node cells taken from mice immunized with horse serum and plated on embryonic fibroblast monolayers. The degranulation capacity of these mast cells and histamine release stimulated by monoclonal IgE antibodies and their antigens were studied. The mast cells were first incubated with either anti‐2,4‐dinitrophenyl (DNP) IgE or with anti‐ovalbumin (OVA) IgE and then with the other IgE to study the ability of one IgE specificity to saturate the receptors and block the ability of the other IgE to bind and evoke degranulation.

Saturation of the receptors and maximal histamine release (86–92%) were obtained within 2–3 h incubation with excess IgE (1–10 μg/ml). No histamine was released after incubation for 3–4 h with the other IgE (0.38–4.0% histamine release). Seventeen days after the excess, unbound, saturating IgE anti‐DNP was washed away, 75% of the histamine was still released after antigen DNP‐bovine serum albumin was added. However, these mast cells were still effectively blocked from sensitization with IgE anti‐OVA (1.71% histamine release).

The blocking could be broken if the second IgE molecules were allowed to stay longer than 4 h in culture. From 10 h onwards, the degranulation capacity steadily increased (from 59.6% histamine release after 10 h to 79.5% after 42 h). In studies with 125I‐labeled IgE, there was a direct correlation between the rate of binding of the second IgE to the mast cells (from 1.7% binding after 3 h to 75% after 48 h) and the increase in degranulation rate with the second antigen (from 2.40% histamine release to 65.4%). In contrast, only slight binding of the 125I‐labeled IgE of the saturating specificity occurred (4.2% after 3 h to 12.7% binding after 48 h).

Incubation of mast cells, previously saturated with 125I‐labeled IgE, with cold IgE of either specificity did not proportionally reduce the cell‐bound label. This suggests that no substitution of IgE molecules on the receptors occurred. When the mast cells saturated with anti‐OVA were incubated with IgE anti‐DNP together with tunicamycin, the development of the degranulation capacity by DNP‐bovine serum albumin was inhibited. The results suggest that IgE molecules of the other specificity stimulated the appearance of new receptors on the mast cell surface.
Identification of a uveitogenic cyanogen bromide peptide of bovine retinal S‐antigen and preparation of a uveitogenic, peptide‐specific T cell line
Authors: Gregerson Dale S. , Obritsch Wesley F. , Fling Steven P.
Published: March 2007

Peptide fragments of bovine retinal S‐antigen produced by cyanogen bromide (CB) digestion have been purified and tested for their ability to induce experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in Lewis rats. Following immunization with the various peptides in complete Freund's adjuvant, one of the peptides, CB123, was found to be potently uveitogenic. A CB123‐specific, class II restricted T helper lymphocyte line (R208) prepared from one of the CB123 peptide‐immunized animals by repeated in vitro selection with purified CB123 was able to transfer severe EAU and EAP to naive rats. Peptides CB36, CB46, CB51 and CB66 were immunogenic as assessed by the presence of antibodies detected in the enzyme‐linked immunosorbent assay and sensitized lymphocytes found in [3H]thymidine incorporation assays using lymphocytes from the peptide‐immunized rats, but they did not induce significant EAU or EAP. Antibodies induced by immunization with the peptides also bound intact S‐antigen, unlike the lymphocytes which were only weakly responsive to S‐antigen. Cross‐reactivity of antibodies and lymphocytes from CB51 and CB66 immune animals suggested that these peptides overlap and contain a common epitope. No measurable immunity to any CB peptide or intact S‐antigen was found in the animals immunized with peptide CB26. Although a T cell line (R17) raised to human S‐antigen was also able to transfer EAU, it was weakly responsive to the CB123 peptide; also, the R208 line did not respond well to human S‐antigen. Since both human and bovine S‐antigen are uveitogenic, these results suggest that the uveitogenic epitope in CB123, which is of bovine origin, is not the same as the uveitogenic epitope of the human antigen.
Isolation of rat immunoglobulin class switch variants of rat‐mouse hybridomas by enzyme‐linked immunosorbent assay and sequential sublining
Authors: Pluschke Gerd , Bordmann Gerard
Published: March 2007

The use of sequential sublining in combination with highly specific and sensitive enzyme‐linked immunosorbent assays for the isolation of spontaneous rat Ig heavy chain class switch variants is described. These methods allowed us to isolate switch variants from mouse‐rat hybridoma lines secreting monoclonal rat antibodies. Switch variants from IgM to IgG2a, from IgG2a or IgG2b to IgE and from IgE to IgA were obtained. Members of the BA1.2 family, which consists of IgG2b, IgE and IgA antibodies are shown to exhibit identical rhamnose‐inhibitable binding to the O18A antigen of Escherichia coli and to the paratope‐associated anti‐idiotypic antibody BA114.
LFA‐1 β‐chain synthesis and degradation in patients with leukocyte‐adhesive proteins deficiency
Authors: Dimanche Marie‐Therèse , Le Deist Franç , Fischer Alain , Arnaout M. Amin , Griscelli Claude , Lisowska‐Grospierre Barbara
Published: March 2007

The defective membrane expression of the adhesive protein family (LFA‐1, Mo1 and p 150, 93) on leukocytes from certain patients with recurrent bacterial infections was shown to be secondary to the absence of synthesis of mature β chain that is common to all three antigens (Springer et al., 1984, Lisowska‐Grospierre et al. 1986). In all patients, studies of β‐chain biosynthesis that lead to this conclusion were performed using the monoclonal anti‐β chain antibody to isolate the β subunit. Since this antibody detects the mature form of β chainonly, the potential presence of a precursor or of an abnormal β chain in the patient's cells could not be tested. the availability of the polyclonal antibody to the purified β subunit allowed us t re‐examine the biosynthesis of the LFA‐1 subunits in 3 affected children. In al 3 patients, the absence of membrane expression of the LFA‐1, CR3 and p150,95 proteins was confirmed. The LFA‐1 α‐chain precursor of 170 kDa was detected in the lysates of PHA blasts of two children, but was not detected in the third. The β‐chain precursor of 85 kDa was isolated by the polyclonal anti‐β chain antiserum from the cytoplasm of phytohemagglutinin and Epstein‐Barr virus‐induced blasts of one patient. The same antibody precipitated some peptides of smaller mol. wt. from the cell lysates of 2 other patients. These results suggest that in this disorder the membrane nonexpression of the adhesive proteins is probably due to the structural abnormality of β chain which, although synthesized, is rapidly degradated.
Antibody diversity in amphibians. Noninbred axolotls used the same unique heavy chain and a limited number of light chains for their anti‐2,4‐dinitrophenyl antibody responses
Authors: Charlemagne Jacques
Published: March 2007

Noninbred axolotls (Ambystoma mexicanum, amphibia, urodela) were immunized with trinitrophenylated sheep red blood cells (TNP‐SRBC) and anti‐2,4‐dinitrophenyl (DNP)/TNP antibodies were individually purified by affinity chromatography. The isolated IgM‐like antibodies were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and isoelectric focusing (IEF) under reducing conditions. The SDS‐PAGE and IEF‐separated heavy (H) and light (L) chains were electroblotted onto nitrocellulose, probed with mouse monoclonal antibodies specific for H or L axolotl Ig chains and stained by a rabbit anti‐mouse Ig horseradish peroxidase conjugate. The specific detection of axolotl anti‐DNP/TNP H chain spectrotypes shows for each of the 14 individually analyzed samples a very similar pattern of 4–5 ordered spaced bands. This suggests that all animals express the same VH chain segment representing the germinal expression of a unique VH gene. When the same analysis was performed starting from a pool of nonimmunized axolotl sera, a low background of natural anti‐DNP antibodies was detected. When analyzed by IEF, the H chains of the pooled anti‐DNP natural antibodies display the same pattern of restricted heterogeneity when compared to the H chain spectrotypes of the individual immune anti‐DNP/TNP antibodies. The specific detection of the axolotl anti‐DNP/TNP L chain spectrotypes indicates at the individual level more heterogeneous and polymorphic patterns compared with H chains, although most animals share the majority of their bands. Our experiments indicate that in axolotl, the production of antibodies to DNP results from the germinal expression of a very limited set of V genes, already expressed as naturally occurring anti‐DNP antibodies before immunization. This seriously restricts the possible extension of the antibody repertoire and perhaps even the nature of antibody “specificity” in this primitive vertebrate.
Induction of an auto‐anti‐IgE response in rats II. Effects on mast cell populations
Authors: Marshall Jean S. , Prout Samantha J. , Jaffery Ghazala , Bell Eric B.
Published: March 2007

Immunization of rats with purified rat IgE myeloma (IR2) induces an IgG class atuoantibody directed specifically against the IgE isotype. This has variable stimulatory effects on the serum IgE concentration in high IgE‐producing BN rats but significantly decreases the serum IgE concentration in the conventional PVG.RT1u strain. We have examined the effects of inducing such an auto‐anti‐ϵ response on mast cell populations, as defined by their staining characteristics in BN rats. A persistent anti‐ϵ response changed the proportions of mast cell types. Those containing highly sulfated, Safranin‐positive granules which equate with cells described as connective tissue mast cells (CTMC) were reduced in number in skin, tongue and bone marrow, whereas the less highly sulfated, Alcian Blue‐positive mast cells were increased in number in these tissues as well as in the gut, a site rich in the so‐called mucosal mast cell. The overall number of mast cells in nonmucosal sites was not significantly changed in anti‐IgE‐producing rats.

The direct effects of anti‐ϵ antiserum on mast cells in vivo were investigated using an immediate skin response (ISR) technique. The IgG component of serum from IR2‐immunized rats, fractionated by high performance liquid chromatography or eluted from Sepharose 4B‐coupled IR2, gave positive ISR in naive rats. The ISR was inhibited by prior incubation of immuno‐purified rat anti‐IR2 with solid‐phase IR2 or with two unrelated IgE myeloma proteins but not with rat IgG. Histological examination confirmed that degranulation of CTMC had occurred at the ISR sites.
Phenotypic identification of suppressor‐effector, suppressor‐amplifier and suppressor‐inducer T cells of B cell differentiation in man
Authors: Kansa Geoffrey S. , Engleman Edgar G.
Published: March 2007

Using monoclonal anti‐Leu 8 antibody to isolate subpopulations of human helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T cells, we have investigated the role of these subpopulations in the regulation of B cell differentiation in the human autologous mixed leukocyte reaction (AMLR). Whereas AMLR‐activated CD8+,Leu8− cells were capable of suppressing fresh AMLR cultures in the absence of fresh CD8+ cells, CD8+, Leu8+ cells suppressed only those cultures containing fresh CD8+ cells. On the other hand, CD8+, Leu8− cells became suppressor cells only when cultured in the presence of CD8+,Leu8+ cells. Finally, the development of CD8+ suppressor cells was dependent on the presence of CD4+,Leu8+ cells; CD4+,Leu8− cells were incapable of acting as suppressor‐inducer cells, but have been shown previously to mediate T cell help for B cell differentiation. Thus, at least 3 phenotypically distinct subsets of T cells interact sequentially to generate suppression of B cell differentiation induced in the AMLR: CD4+,Leu8+ suppressor/inducer cells, CD8+,Leu8+ suppressor‐amplifier cells and CD8+Leu8− suppressor‐effector cells.
Thymic immune response gene function in radiation chimeras reconstituted with purified hemopoietic stem cells
Authors: Kast W. Martin , Voordouw Arie C. , Leupers Tine , Visser Jan W. M. , Melief Cornelis J. M.
Published: March 2007

Thymectomized (C57BL/6[B6] × bm1)F1 mice and thymectomized (B6 × bm12)F1 mice were engrafted with neonatal parental thymus of either B6 type [H‐2b mouse, Sendai virus cytotoxic T cell (Tc) responder] or bm1 type (H‐2Kb mutant, Sendai virus Tc nonresponder) and B6 type (H‐Y Tc responder) or bm12 type (H‐2 I‐Ab mutant, H‐Y Tc nonresponder), respectively. All mice were irradiated and reconstituted with highly purified syngeneic pluripotent hemopoietic stem cells. All types of thymus engraftment resulted in a restored T cell immunocompetence. The Tc reaction to Sendai virus in (B6 × bm1)F1 mice engrafted with both responder type B6 and nonresponder, type bm1 neonatal thymus allowed maturation of Sendai‐specific, H‐2Kb‐restricted Tc. For the Tc reaction to H‐Y, only responder type B6 thymus restored the Tc response, whereas this was not achieved with nonresponder type bm12 thymuses. We conclude from this study that in this radiation stem cell chimera system the radioresistant component of the thymus dictates major histocompatibility complex (MHC) specificity and immune response phenotype of T cells restricted to class II MHC molecules but not of T cells restricted to class I MHC molecules.
In vitro studies on human IgD I. Sources and characteristics of “externalized” IgD in tonsil lymphocyte cultures
Authors: Litwin Stephen D. , Zehr Bradley D.
Published: March 2007

As part of a broader analysis into the function of IgD, and especially into the role of human IgD‐secreting cells, fresh human tonsil lymphocyte cultures were analyzed. The goals were to define the origins of “externalized” supernatant IgD and to examine its relationship to spontaneous Ig secretion of IgG, IgM and IgA. Assayable IgD was found by 24 h in cultures without added mitogens. “Externalized” IgD comprised a small fraction (1.6–1.8%) of supernatant Ig when compared to the fraction of IgD‐containing cells (≈ 10%): IgD appears to disappear rapidly in vitro. Initial experiments employing irradiation and chemical inhibitors of protein synthesis suggested that “externalized” IgD was produced de novo during culture rather than representing preformed or cytophilic Ig. Most significantly, tonsil cultures with higher IgD values (> 20 ng IgD/106 cells, day 7) showed convincing evidence that secreted IgD was a major source for “externalized” IgD. This evidence included (a) the amount of “externalized” IgD depended on viable culture conditions; (b) radioincorporated 35S appeared as a visible IgD band on sodium dodecyl sulfate‐polyacrylamidegel electrophoresis (SDS‐PAGE) gels; (c) “higher” IgD cultures showed increased numbers of IgD‐Ig‐containing plasma cells, and IgD,λ in excess of IgD, x; both events are associated with IgD secretion. In cultures producing high amounts of IgD, IgD secretion appeared to be spontaneous rather than induced and was independent of the polyclonal spontaneous Ig secretion of other Ig isotypes. Low titers of inhibitable IgD isotype anti‐phosphorylcholine antibodies but not IgD isotype anti‐polyribotol phosphate antibodies were present in tonsil supernatants: both were present in matched sera.

Other laboratories have directed attention to the striking and possibly selective participation of human tonsil lymphocytes in IgD synthesis using other approaches. Present results supplement and expand earlier data and support the practical value of analysis of short‐term cultured tonsil lymphocytes. Differentiation to IgD‐secreting cells is suggested to be an active, underestimated and putatively an important part of the immune response in the human tonsil.
In vitro studies on human IgD II. IgD‐secreting cells preferentially elaborate IgD, λ molecules
Authors: Litwin Stephen D. , Zehr Bradley D.
Published: March 2007

The “IgD paradox” describes the unexpected finding that, despite a predominance of kappa (x) light (L) chains on the surface of IgD+ human B cells, the majority of monoclonal IgD proteins are of lambda (λ) type. This potentially informative phenomenon appears to be based on a preferential association between delta (δ) heavy (H) and λ L chains of IgD‐secreting cells. The current studies analyze the phenomenon in vitro. Initial assays on tonsil “spent” supernatants showed that the higher IgD‐“externalizing” cultures displayed progressively elevated IgD,λ/x ratios due to parallel shifts in total IgD and IgD,λ while IgD,x remained stable. The cells of the same higher‐IgD cultures demonstrated that individual IgD‐containing cells had a predominance of λ chains in contrast to IgM‐containing cells of the same cultures using fluorochrome‐antibody double staining. Certain tonsil culture IgD‐containing cells remarkably appeared to have exclusively λ chain. These same tonsils show evidence of IgD secretion (Litwin, S. D. and Zehr, B. D., Eur. J. Immunol. 1987. 17: 483); thus, these data align preferential δ‐λ chain association with IgD‐secreting normal tonsil B cells in vitro and emphasize the usefulness of IgD,λ/x ratio in monitoring IgD secretion. In another approach the relationship between the cellular location of IgD and the preferential δ‐λ chain association was studied using Triton X‐114‐partitioned cell lysates. IgD,λ/x ratios were one or less in the detergent phase (membrane‐enriched fraction) consistent with expected IgD,x predominance in membrane IgD. In contrast, the aqueous phase (intracellular‐enriched fraction) of IgD‐secreting cultures had 2–4 times higher supernatant IgD,λ/x ratios. The restriction of high IgD,λ/x ratios to intracellular fractions and supernatants of IgD‐secreting cultured cells parallels the predicted distribution of secretory IgD. In sera studies, the correlation between total IgD and IgD,λ/x ratio values was consistent with secreted sera IgD showing preferential λ chain expression. It was concluded that the phenomenon of δ‐λ chain preferential association is (a) expressed in vitro as well as in vivo; (b) a property of normal, nonmalignant human IgD‐secreting B cells; and (c) closely related to the secretory form of IgD. In certain cultures, the δ‐λ chain preference was so striking as to imply limited heterogeneity of the IgD immune response.
Murine lupus anti‐DNA antibodies cross‐react with the hapten (4‐hydroxy‐5‐iodo‐3‐nitrophenyl)acetyl, but immunization‐induced anti‐DNA antibodies do not
Authors: Zouali Moncef , Migliorini Paola , Stollar David B.
Published: March 2007

The antigen‐binding selectivity of 2 sets of anti‐DNA antibodies from autoimmune mice and from normal mice was examined. Eighteen affinity‐purified anti‐DNA autoantibodies from MRL‐lpr/lpr mice were examined for binding to the haptens azoben‐zenearsonate, phosphorylcholine, (4‐hydroxy‐3‐nitrophenyl)acetyl and (4‐hydroxy‐5‐iodo‐3‐nitrophenyl)acetyl (NIP). Five of these autoantibodies bound to NIP‐protein conjugates. In contrast, none of 12 monoclonal antibodies to single‐stranded DNA or left‐handed Z‐DNA induced by immunization of BALB/c and C57BL/6 mice with nucleic acid antigens reacted with the tested haptens.

In a reciprocal test of the relationship between anti‐DNA and anti‐NIP binding, we examined 24 monoclonal antibodies to NIP, from various strains of mice, for binding to DNA. One such antibody from a BALB/c mouse also bound to DNA. These results are discussed in the context of the mechanisms underlying autoantibody hyperproduction.
Prevention of experimental autoimmune uveoretinitis by active immunization with autoantigen‐specific monoclonal antibodies
Authors: De Kozak Yvonne , Mirshahi Massoud , Boucheix Claude , Faure Jean‐Pierre
Published: March 2007

Preimmunization of Lewis rats with anti‐S antigen (S‐Ag) monoclonal antibodies (mAb) led to protection against experimental autoimmune uveoretinitis (EAU) induced by this retinal autoantigen. High titers of anti‐idiotypic (anti‐Id) antibodies were raised against three mouse mAb, S2D2 (IgG2b), S6H8 (IgG2a) and S7D6 (IgG1), directed at S‐Ag. An almost complete prevention was observed in S2D2 mAb‐immunized animals while a partial protection was achieved with S6H8 and S7D6 mAb. No detectable anti‐Id antibody nor disease prevention was observed in rats immunized with the mAb S9E2 (IgG2a) which only recognizes bovine and sheep S‐Ag, or with control mAb of the same isotypes irrelevant to S‐Ag. The mAb treatment did not modify the level of the whole polyclonal antibody response to S‐Ag. These results suggest an important role in the pathogenesis of EAU for the epitopes recognized by S2D2‐S6H8 and S7D6 in the S‐Ag molecule. The success of anti‐Id immunization for autoimmune disease suppression may depend on the identification of relevant epitopes.
Differentiation in the murine B cell lymphoma I.29: individual μ+ clones may be induced by lipopolysaccharide to both IgM secretion and isotype switching
Authors: Alberini Cristina , Biassoni Roberto , Deambrosis Sandro , Vismara Daniela , Sitia Roberto
Published: March 2007

Cells from the monoclonal B cell lymphoma I.29 expressing surface IgM (μ+) are capable of differentiating in vitro to IgM secretion and of switching to IgA or IgE production in response to lipopolysaccharide (LPS) stimulation. To determine whether a single μ+ B cell is capable of undertaking both differentiative pathways (isotype switch and plasma cell differentiation) I.29 μ+ cells were cloned by limiting dilution and a panel of clones were analyzed by immunofluorescence, endogeneous labeling and Northern blotting. While 100% of the clones could differentiate toward IgM secretion, only a proportion of them (> 70%) also switched to IgA and/or IgE production. Certain clones switched preferentially to a specific isotype. Taken together with the observation that Cγ genes were never the target of switching in our experiments, these data suggest that individual μ+ clones from the I.29 lymphoma are “precommitted” as for their switching potentials.

The subclones that showed a high frequency of switching to IgA transcribed the germ line Cα gene(s), suggesting a role for chromatin structure in determining the isotype switch specificity. Switch variant clones expressing either IgA or IgE on the cell surface were isolated and found capable of further differentiating toward Ig secretion in response to LPS. On the contrary, we could not induce switch to IgA in IgE‐producing cells. Unlike μ+ and α+ cells, all the switch variant clones expressing IgE tested by endogenous labeling constitutively secreted large amounts of IgE in the supernatants even in the absence of LPS stimulation.
Comparison of the effects of 1,25‐dihydroxyvitamin D3 on T lymphocyte subpopulations
Authors: Rigby William F. C. , Yirinec Bruce , Oldershaw Robert L. , Fanger Michael W.
Published: March 2007

Previous studies have demonstrated that 1,25‐dihydroxyvitamin D3 (calcitriol) is a potent inhibitor of human T lymphocyte proliferation. It has been reported that only CD4+ cells are sensitive to the anti‐proliferative action of calcitriol. To further evaluate this observation, we first performed cell cycle analysis of unfractionated phytohemagglutinin‐stimulated peripheral blood mononuclear cells (PBMC) in the absence or presence of calcitriol. The CD4/CD8 ratio was similar between control and treated cells for each phase of cycle (G0→G1, S, G2 + M), suggesting that calcitriol did not selectively block proliferation of either T cell subpopulation. Secondly, the growth‐inhibitory activity of calcitriol on PBMC selectively depleted of either CD4+ or CD8+ cells was comparable to that observed with unfractionated PBMC. Furthermore, the induction of transferrin receptors was inhibited by calcitriol to a comparable degree in each T cell subset, suggesting that equivalent inhibition of transition into late G1 was observed. Finally, calcitriol inhibited the proliferation of highly purified T cell subsets (> 99% pure) to equivalent degrees. These data suggest that T cell subsets defined by either the CD4 or by the CD8 antigen are both sensitive to the growth inhibitory effects of calcitriol.
Hybrid antibody‐mediated lysis of virus‐infected cells
Authors: Staerz Uwe D. , Yewdell Jonathan W. , Bevan Michael J.
Published: March 2007

Previously, cytotoxic T lymphocytes (CTL) had only been focused by hybrid antibodies to normal, cell‐surface proteins and haptenated surface structures. In this report, we extend the application of this technology to mediate lysing of virus‐infected cells by nonspecific CTL. Heteroconjugated antibodies between the anti‐T cell antigen receptor antibody, F23.1, and monoclonal antibodies against either the hemagglutinin or nucleoprotein of the influenza virus PR/8 were constructed. We show in the present report that these bispecific constructs can target virus‐infected cells for lysis according to the specificity of the virus‐protein reactive monoclonal antibodies. Furthermore, even a virus protein that is only expressed in small quantities on the cell surface, such as nucleoprotein, can be exploited as target structure for heteroconjugated antibodies. These studies show that hybrid antibodies can focus a CTL response on virus‐infected cells which might in the future be used to mount an immune response in situations without sufficient normal cellular defense.
Constitutive production of B cell differentiation factor‐like activity by human T and B cell lines
Authors: Kanowith‐Klein Susan , Saxon Andrew , Uittenbogaart Christel H.
Published: March 2007

A lymphokine demonstrating human B cell differentiation factor (BCDF)‐like activity was isolated from immature (MOLT‐4f, CCRF‐CEM and CCRF‐HSDB‐2) and mature (HUT‐78) malignant human T lymphoid cell lines and from human B lymphoblastoid cell lines (BJAB and ALL‐7031‐B). All the cell lines were grown long term in serum‐free medium. This BCDF‐like activity has a molecular mass in the range of 40–60 kDa and stimulates immunoglobulin synthesis of cell lines capable of producing IgA (GM‐1056), IgG (GM‐1500 and CESS) and IgM (CBL#3). It was not produced by a myeloid cell line. We were only able to identify the differentiation activity produced by the T and B cell lines by using appropriate molecular mass fractions from the serum‐free medium as controls. This BCDF‐like activity is different from that of the human BCDF so far described as it has a higher molecular mass and is constitutively produced by malignant T lymphoid cell lines which are human T cell leukemia virus‐I negative and by B lymphoblastoid cell lines.
Isotypic pattern of the polyclonal B cell response during primary infection by Plasmodium chabaudi and in immune‐protected mice
Authors: Falanga Pierre Barthélémy , D'imperio Lima Maria Regina , Coutinho Antonio , Da Silva Luiz Pereira
Published: March 2007

The primary infection by P. chabaudi induces an increase of the numbers of splenic immunoglobulin (Ig)‐secreting B cells in both athymic and euthymic BALB/c mice. The isotypic pattern of the polyclonal response is restricted only in euthymic mice where IgG2a, IgG2b and IgM plaque‐forming cells (PFC) predominate. In immunized animals, protected against a parasite challenge, the isotypic pattern of splenic PFC is completely different, the IgG1 and IgM isotypes constituting the main part of the response. Reinoculation of immune‐protected animals induces a PFC response which is dose dependent and accentuates the characteristic isotypic profile of the immune‐protected mice.
The induction of class I HLA by interferon‐α is independent of the cell cycle, but the expression is enhanced by a G1/S block
Authors: Erusalimsky Jorge D. , Gilmore David , Milstein Cesar
Published: March 2007

The induction of class I HLA expression by interferon‐α (IFN‐α) was studied in lymphoid cells arrested or traversing different stages of the cell cycle. Exponential cultures of MOLT‐4 cells and the MOLT‐4 cell variant YHHH were treated with the cell cycle inhibitors aphidicolin and colcemid to obtain cell populations arrested in G1/S and G2/M, respectively, and also cells traversing from S to M and vice versa. Cytofluorimetry with the monoclonal antibody YTH/76.3 (which specifically detects those class I molecules which are most susceptible to IFN‐α induction) was used to quantitate the class I HLA response to IFN‐α. The results showed that the response to IFN‐α is not restricted to a given stage of the cell cycle. These studies also revealed that when the cells were arrested at G1/S, the absolute level of class I HLA expression was enhanced 2–3‐fold, both in the presence or absence of either IFN‐α or IFN‐γ. Therefore, even when absolute levels changed, the ratio of IFN‐induced expression to basal expression remained constant at all cell cycle stages. The level of expression of another surface antigen (the CD1 antigen HTA‐1) was not affected by the G1/S block. The results were confirmed by dot blot hybridization of poly (A)+ RNA using cDNA‐specific probes. These findings suggest that the effect of IFN‐α is continuous throughout the cell cycle but that a G1‐dependent event determines the extent of class I HLA expression, and leads to a synergistic superinduction by IFN in G1/S‐arrested cells.
Modulation of neutrophil expression of C3b receptors (CR1) by soluble monomeric human C3b
Authors: Porteu Françoise , Mir Amparo , Halbwachs‐Mecarelli Lise
Published: March 2007

Upon incubation at 37°C with purified human C3b (500 μg/ml), polymorphonuclear neutrophils (PMN) were found to express up to 50% more C3b receptors (CR1) than PMN incubated with buffer alone. This up‐regulation of CR1, assessed by the binding of radiolabeled CR1‐specific monoclonal antibody, was dependent on the dose of C3b, occurred within 10–20 min and was stable for at least 90 min. PMN incubated with C3b also demonstrated enhanced CR1‐dependent binding functions, such as EC3b rosette formation and phagocytosis of EIgGC3b particles. C3b at a concentration of 500 μg/ml induced up to 90% increase in the attachment or the phagocytic index. However, CR1 remained unable to promote phagocytosis of EC3b intermediates. Fc receptor‐mediated functions were unaffected by the treatment with C3b. The active factor was characterized as monomeric C3b and, in particular, shown to be distinct from C5a. C3b purified by anion‐exchange fast protein liquid chromatography on a Mono Q column or eluted from a monoclonal anti‐C3b‐Sepharose retained its modulating activity, while native C3 or C3 fragments such as iC3b, C3c or C3d,g were ineffective.
T cell receptor β chain gene rearrangements and Vβ gene usage in horse cytochrome c‐specific T cell hybridomas
Authors: Baumhüter Susanne , Fasel Nicolas , Corradin Giampietro , Guardiola John
Published: March 2007

Comparison of T cell receptor α and β‐chain genes in murine major histocompatibility complex (MHC) class I and class II‐restricted T cell clones and hybridomas recognizing different antigens indicates that no simple correlation exists between the observed antigen/MHC specificity and the expression of certain α and β‐chain heterodimers. We have attempted to establish a possible correlation by analyzing T cell receptor β chain gene rearrangements and Vβ gene usage in five T cell hybridomas with identical antigen/MHC specificity and another hybridoma recognizing a different antigenic determinant in association with the same restriction molecule. We report here that in each of the five clones a uniquely rearranged β chain gene is expressed in combination with at least two different Vβ gene segments. The presence of the differently rearranged T cell receptor β chain genes correlated with the finding of distinct fine specificity pattern of antigen recognition in each of the hybridomas. Interestingly, two hybridomas specific for different epitopes showed identical β chain D‐J rearrangements indicating that the differences might be encoded by the α chain gene or/and the Vβ gene element.
Effect of stereospecificity of chemically synthesized lipid A‐subunit analogues GLA‐27 and GLA‐40 on the expression of immunopharmacological activities
Authors: Kumazawa Yoshio , Ikeda Satoru , Takimoto Hiroaki , Nishimura Chiaki , Nakatsuka Mitsunobu , Homma J. Yuzuru , Yamamoto Akihiro , Kiso Makoto , Hasegawa Akira
Published: March 2007

Tumor necrosis factor (TNF)‐inducing, mitogenic, polyclonal B cell activation (PBA), macrophage activation and antiviral activities of chemically synthesized lipid A‐subunit analogues, GLA‐27 and GLA‐40, were investigated. The structure of GLA‐27 comprises 4‐O‐phosphono‐D‐glucosamine carrying tetradecanoyl and 3‐tetradecanoyloxytetradecanoyl [C14‐O‐(C14)] groups as the 3‐O‐ and 2‐N‐acyl substituents, respectively. GLA‐40 is a 1‐deoxy compound of GLA‐27. The activities of stereoisomers, (R) and (S) forms at the C3 position of the C14‐O‐(C14) group, of both compounds were also investigated. TNF‐inducing activity of the (S) isomers of GLA‐27 and GLA‐40 was stronger than that of the (R) isomers while the (R) isomers exhibited stronger mitogenic and PBA activities than the (S) isomers. With respect to macrophage activation such as phagocytosis, acid phosphatase and N‐acetyl‐β‐D‐glucosaminidase activity as cellular lysosomal enzymes and cytostasis, peritoneal macrophages obtained from mice admistered i.p. with test samples showed significant activities. Among stereoisomers of GLA‐27, the (R) isomer exhibited somewhat stronger phagocytic and lysosomal enzyme activities than those of the (S) isomer while there was no appreciable difference in the activities between the isomers of GLA‐40. Significant cytostasis‐inducing activity was observed in stereoisomers tested. All of the isomers showed remarkable antiviral activity against vaccinia virus.
DNA fragmentation and cytotoxicity caused by tumor necrosis factor is enhanced by interferon‐γ
Authors: Dealtry Gillian B. , Naylor M. Stuart , Fiers Walter , Balkwill Frances R.
Published: March 2007

Recombinant human tumor necrosis factor (rhuTNF) induced DNA fragmentation in sensitive cell lines. This fragmentation was similar to that caused by lymphotoxincontaining cytotoxic T cell culture supernatants. The percentage of DNA cleaved correlated with the degree of cell growth inhibition shown by individual cell lines. DNA fragmentation was first seen after 12 h treatment, and increased slowly with time. The presence of 100 μM ZnSO4 inhibited the rhuTNF‐induced DNA cleavage in MCF‐7 cells. Recombinant interferon‐γ (rhuIFN‐γ) did not induce DNA cleavage, although it reduced the growth of all the cell lines used in this study. However, it interacted with rhuTNF to produce a doubling in the percentage of DNA fragmentation, and increased cytotoxicity in rhuTNF‐sensitive cell lines. Pretreatment with rhuIFN‐γ for 1 h prior to rhuTNF treatment also enhanced DNA fragmentation and cell killing.
Inhibition of interleukin 3 function by a fragment of the third component of complement
Authors: Faßbender Birgit , Schmitt Edgar , Bitter‐Suermann Dieter , Hadding Ulrich
Published: March 2007

A C3d‐like (C3d‐l) fragment of 33 kDa was isolated and its biological activity studied. The fragment was generated from guinea pig C3b by porcine pancreas kallikrein and purified by fast protein liquid chromatography. The C3d‐like fragment inhibited interleukin (IL) 2‐dependent T lymphocyte proliferation. The suppressive activity of the described C3d‐l fragment was not restricted to lymphocytes as targets but inhibited in addition the proliferation of a nonlymphocyte mast cell line which was strictly IL 3‐dependent in its proliferative capacity. Kinetic studies implied early stages of cellular proliferation to be influenced. Furthermore, the C3d‐l fragment was not only an inhibitor of cellular proliferation but was also a potent inducer of leukocytosis.
Enhanced frequency of mitogen‐responsive T cell precursors in old mice injected with thymosin α1
Authors: Frasca Daniela , Adorini Luciano , Doria Gino
Published: March 2007

Injection of old mice with thymosin α1, a synthetic peptide consisting of 28 amino acid residues and exhibiting thymic hormone‐like activity, increases the splenic frequency of T cell precursors. Young (3‐month‐old) or old (19–20‐month‐old) mice received a single i.p. injection of thymosin α1 or of an equimolar amount of the N14 (N‐terminal amino acid residues 1–14) or C14 (C‐terminal amino acid residues 15–28) synthetic fragment of the thymosin α1 molecule and their spleen cells were assayed 3 days later under limiting dilution conditions to assess the frequency of mitogen‐responsive and interleukin 2‐producing T cells. Injection of thymosin α1 or of its N14 fragment increases the frequency of responsive T lymphocytes in old, but not in young mice whereas injection of the C14 fragment has no demonstrable effect in either young or old mice. These data are consistent with our previous observation that the biological activity of thymosin α1 is restricted to the N‐terminal half of the molecule and suggest that this peptide amplifies the pool of mitogen‐responsive and interleukin 2‐producing T cells in immunodeficient old mice.
Monoclonal anti‐allotype antibody towards BALB/c IgM Analysis of specificity and site of a V‐C crossover in recombinant strain BALB‐Igh‐Va/Igh‐Cb
Authors: Schüppel Ralph , Wilke Joachim , Weiler Eberhardt
Published: March 2007

Polyclonal IgM obtained by fractionation of a BALB/c serum pool was used as the immunogen for C57BL/6 mice. Draining lymph nodes from selected animals donated cells for fusion with myeloma Sp‐2/0. Fifteen hybridomas were productive, and one (RS‐3.1) was cloned and the affinity‐purified product analyzed for its reactivity pattern by MOPC 104E‐enzyme‐linked immunosorbent assay inhibition. Among five BALB/c myelomas only TEPC 183 (IgM) was active, not those belonging to other Ig classes. Among normal sera from 8 mouse strains only those of BALB/c, DBA/2J and CBA/J showed inhibition.

The recombinant strain BALB‐Igh‐Va/Igh‐Cb did not react, which shows that its Cμ stems from parental strain C57BL/6, and that therefore the recombination event had occurred 5′ of this gene.
An antigen‐independent physiological activation pathway for L3T4+ T lymphocytes
Authors: Germann Tieno , Huhn Helga , Zimmermann Frank , Rüde Erwin
Published: March 2007

The data presented in this report describe an antigen‐independent activation pathway leading to reinduction of proliferation of class II major histocompatibility complex (MHC)‐restricted murine T cell lines that after previous antigen‐specific stimulation reverted to a resting state. Antigen‐independent proliferation and interleukin 2 (IL 2)‐receptor expression occur in the presence of splenic accessory cells, exogenous IL 2 and a soluble factor(s) provisionally termed T cell‐stimulating factor(s) (TSF). Each of these components is essential for inducing growth. TSF is found in the supernatant of an autoreactive T cell line upon stimulation with syngeneic accessory cells. Neither TSF nor accessory cells can be replaced by IL1 and by some other cytokines. Monoclonal antibodies against class IIMHC molecules, the T cell receptor and L3T4 do not block this antigen‐independent stimulation. This demonstrates that the function of the accessory cell in this system is not MHC restricted and that the T cell receptor is also not involved. Furthermore, it is suggested that the blocking of L3T4 molecules by antibody will mediate a negative signal only if T cells are triggered via their antigen receptors.
Specific helper T cell reactivity against autologous erythrocytes implies that self tolerance need not depend on clonal deletion
Authors: Hooper D. Craig , Taylor Roger B.
Published: March 2007

Using a culture system which supports primary T cell proliferative responses to various antigens we have detected mouse red blood cell (RBC)‐reactive T cells in lymphoid tissues from untreated mice. The release of significant amounts of interleukin 2 (IL2) indicates that T helper (or helper/inducer) cells are activated by stimulation with RBC. Upon restimulation in vitro these cells proliferate specifically against mouse RBC with the kinetics and magnitude characteristic of a secondary response. Since autologous RBC are tolerated in vivo in spite of the presence of such specifically reactive T helper cells, these findings imply that self tolerance, even to certain nonse‐questered antigens, may depend largely on regulatory mechanisms rather than on clonal deletion or inactivation.