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T Diamantstein

Publications and source records attributed to T Diamantstein.

At least 55 records · Page 3Linked to original sources
PubMed1991

Generation of competitor DNA fragments for quantitative PCR.

A convenient and generally applicable method for the generation of competitor DNA fragments for quantitative PCR is described. Using mouse-specific primers, fragments are amplified from DNA of an evolutionarily distantly related species under low-stringency annealing conditions. Because these artificially created fragments contain the mouse primer specific ends, they can be used for the quantification of the mouse DNA amplified by these primers. Competitor DNA fragments that differ in size from the corresponding mouse DNA are selected to distinguish both fragments visually by gel electrophoresis. Competitor DNA fragments were generated for mouse beta-actin, interleukin-1, and tumor necrosis factor (TNF). Co-amplification of beta-actin cDNA for adjustment of equal amounts of input cDNA and subsequently TNF cDNA from lipopolysaccharide (LPS)-activated and nonactivated spleen cells with serial dilutions of the respective competitor DNA fragments allowed a semiquantitative comparison of the ratio of TNF mRNA present in both cDNA samples. Under certain conditions, the competitor DNA fragments can be used to determine the approximate molar concentration of a mRNA.

Animals↗
PubMed1991

[Immunorestitutive action of hydrolysates and ultrafiltrates of bovine spleen].

Investigations with different spleen extracts of varied composition and purity have shown immunomodulatory effects. In the present study the in vivo effect of a bovine spleen hydrolysate (Prosplen) on a radiation-induced immunodeficiency model is demonstrated. Intraperitoneal application of 1 mg/g bw hydrolysate 3/times a week caused a highly significant acceleration of the restitution of the humoral and cell-mediated immunoreactivity towards sheep erythrocytes in sublethally (600 cGy) radiated mice: plaque-forming cells in the spleen, hemagglutination, hemolysistiter of the serum, delayed type of hypersensitivity = DTH reaction. The immunoreactivity of the treated groups reached normal levels within 6-8 weeks, whereas in the groups receiving placebo this was observed after 10 weeks at the earliest. Cytofluorometric investigations revealed an accelerated restitution of the B-lymphocyte fraction in the spleen. Mice that was not irradiated showed no effect after treatment with the hydrolysate. Similar results were obtained using an ultrafiltrate of the hydrolysate.

Adjuvants, Immunologic↗
PubMed1990

DNA rearrangement and constitutive expression of the interleukin 6 gene in a mouse plasmacytoma.

To study the potential involvement of IL-6 in the development of plasmacytomas, a number of plasmacytoma lines were analyzed for alterations in the IL-6 locus. A DNA rearrangement due to the insertion of an intracisternal A particle retrotransposon 18 bp 5' of the transcriptional start site was detected in the cell line MPC11. The IL-6 gene is constitutively expressed in MPC11, suggesting the involvement of IL-6 in the development of certain myeloma/plasmacytomas according to the "autocrine growth hypothesis".

Animals↗
PubMed1990

Retroviral interleukin 4 gene transfer into an interleukin 4-dependent cell line results in autocrine growth but not in tumorigenicity.

A retroviral vector has been constructed containing the mouse interleukin 4 (IL 4) gene under the transcriptional control of the thymidine kinase promoter. Infection of the IL 4-dependent T cell line CT4S by the thymidine kinase IL 4 construct resulted in factor-independent growth. The infected cells grow as a consequence of continuous consumption of the endogenously produced IL 4. Clones were established secreting different amounts of IL 4 but none of them was capable of growing in vivo. The lack of correlation between factor-independent growth and tumorigenicity distinguishes IL 4 from other growth factors and could reflect the recently reported activity of IL 4 to suppress tumor growth in vivo.

Animals↗
PubMed1990

Retroviral interleukin 5 gene transfer into interleukin 5-dependent growing cell lines results in autocrine growth and tumorigenicity.

Two interleukin 5 (IL5)-specific retroviral expression vectors have been constructed containing the neomycin gene as selectable marker and either the mouse IL5 cDNA region or the rat genomic IL5 gene under the control of the thymidine kinase promoter. High viral titer supernatants derived from the transfected or infected packaging cell line psi 2 were used to infect the two cell lines B13 and T88M whose growth is dependent on exogenous IL 5. Infection resulted in G418 resistance and IL 5-independent growth with a high frequency. Clones were established which secrete between 2 and greater than 1000 U IL5. The proliferation of the IL5 autocrine growing cells could be inhibited by an antibody directed against the IL5 receptor indicating that they grow as a result of the endogenously produced IL5. Regardless of the amount of IL5 they produced, all of the clones were highly tumorigenic in nucle mice. The phenotype of the tumors was indistinguishable from that of the injected cells. T88M or B13 cells infected with a control virus neither produced IL5, nor became factor independent, nor produced tumors. Together, the IL5 gene transfer and expression into IL5-dependent growing cells are in accordance with the "autocrine growth" hypothesis and contrast analogous experiments with IL4.

Animals↗
PubMed1990

High affinity IL-2 receptors on a Hodgkin's derived cell line.

Hodgkin and Sternberg Reed (H and SR) cells, the putative malignant cells of Hodgkin's disease carry regularly T-cell activation antigens, like CD30 and CD25 (low affinity IL-2 receptor). We have investigated the Hodgkin cell line L540, bearing characteristic markers of H and SR cells for its expression of the low affinity IL-2 receptor (IL-2R) and for IL-2. Expression of the low affinity IL-2R was found on mRNA level, by detection of specific 3.5 kb and 1.4 kb mRNA and on the protein level by immunoprecipitation of a 55,000 mol. wt molecule from detergent extracts of surface iodinated cells, however IL-2 specific mRNA was not detected. Scatchard plot analysis revealed the presence of 2 x 10(3) high affinity IL-2Rs. Crosslinking experiments directly demonstrated the high affinity IL-2R to consist of the 55,000 mol.wt light chain (L), and the 70/75,000 (H1/H2) heavy chains. IL-2 was rapidly internalized by these receptors, suggesting that they can be functional. The expression of functional IL-2Rs might be involved in induction or differentiation of Hodgkin's disease.

Fluorescent Antibody Technique↗
PubMed1990

Lack of tumorigenicity of interleukin 4 autocrine growing cells seems related to the anti-tumor function of interleukin 4.

Recently, the failure of interleukin 4 (IL4) autocrine growing CT4S cells to grow in vivo has been demonstrated. Because it could not be excluded that the cells produce insufficient amounts of IL4 to support their growth in vivo, subclones were established which are unresponsive to exogenous IL4 and therefore have acquired full growth autonomy. From the fact that the subclones likewise did not give rise to tumors when injected into nude mice, one may conclude that the IL4 production of autocrine growing CT4S prevents their growth in vivo. To test this hypothesis, a retroviral vector containing the IL4 gene under the control of the immunoglobulin heavy chain (Igh) enhancer/promoter was constructed and used to infect the myeloma cell line J558L. An IL4 producing clone was established (J558L-XEPIL4) and the tumor progression in comparison to the parental clone J558L was monitored in nude mice. The IL4 production significantly delayed the growth of J558L-XEPIL4 in vivo. Tumor suppression was much more evident when J558L-XEPIL4 cells were injected into syngeneic BALB/c mice. These results may explain why autocrine growing CT4S do not grow in vivo and suggest the involvement of functional T lymphocytes in the effectiveness of the host dependent anti-tumor action of IL4.

Animals↗
PubMed1990

The effect of T cell subset depletion on autoimmune thyroiditis in the Buffalo strain rat.

The aim of these experiments was to analyse the functional roles of phenotypically defined T cells from Buffalo strain rats with immunisation or neonatal thymectomy-induced autoimmune thyroiditis. Rats were depleted either of CD8-positive T cells by administration of the Ox8 monoclonal antibody or of activated T cells by administration of low-dose cyclosporin A (Cs A) with an anti-IL-2 receptor monoclonal antibody (ART-18), and the effects on subsequent disease assessed. Even though animals were not completely depleted of Ox8 cells, immunisation-induced thyroiditis was enhanced by Ox8 treatment, whereas thyroglobulin antibodies were reduced compared with controls. Subtherapeutic doses of either Cs A or ART-18 alone had little effect on thymectomy-induced thyroiditis, in contrast to Cs A and ART-18 in conjunction, which prevented disease developing. These results suggest important roles for CD8-positive and IL-2 receptor-bearing T cells in experimental autoimmune thyroiditis.

Animals↗
PubMed1990

Evolutionary aspects, structure, and expression of the rat interleukin 4 gene.

The rat interleukin 4 (IL 4) gene has been isolated from a genomic lambda phage library by cross-hybridization to the mouse IL 4 cDNA. Like the mouse and human counterparts, it exists as a single copy gene in the genome and consists of four exons. The overall structure of the IL 4 locus seems highly conserved. This is indicated by the low degree of restriction fragment length polymorphism in a number of laboratory and wild mice and by the conservation of the intron size between human, rat, and mouse IL 4 genes. Furthermore, evolutionary conserved elements are the promoter region, the position of cysteine residues and sequence motifs in the 3' untranslated regions that are believed to be involved in destabilization of the mRNA. In contrast, the predicted amino acid sequence of the rat IL 4 gene shows low homology (57%) with the mouse homologue. The divergence between mouse and rat IL 4 genes is even more pronounced in the carboxy-terminal region (47% homology in the last 68 amino acids). The ratio between replacement and silent mutations in the IL 4 genes of different species suggests a complex pattern of selective forces acting on the IL 4 gene, which includes both selection against and for amino acid substitutions in individual positions. The functional identity with IL 4 has been confirmed by expression of the gene and the demonstration of the ability to induce MHC class II antigen expression on spleen cells.

Amino Acid Sequence↗
PubMed1990

Different lymphocyte subset distribution within "insulitis" islets of normoglycaemic and prediabetic BB/OK rats of similar age.

The fact that insulitis occurs also in normoglycaemic BB rats led us to investigate the phenotypes of lymphocytes invading the pancreatic islets of prediabetic BB/OK rats in comparison to age- and sex-matched normoglycaemic animals in a retrospective analysis. By using a panel of monoclonal antibodies we investigated the number of pan T-cells, T-helper cells, cytotoxic T-cells and NK-cells and determined the number of activated cells by measurement of class I, class II and IL-2 receptor positive cells. The bound primary antibodies were visualized using the APAAP-technique. The prediabetic rats showed a significantly decreased pancreatic insulin content which was drastically reduced at diagnosis of diabetes. This was accompanied by reduction of the B-cell volume density. The prediabetic as well as the long-term normoglycaemic BB rats showed an accumulation of mononuclear cells (all phenotypes investigated) within the pancreatic islets. Concerning the phenotypes of infiltrating cells there was no qualitative difference between long-term normoglycaemic and prediabetic rats but quantitatively an enhanced amount of W3/25+, OX-8+, OX-6+ and ART-18+ cells could be observed in the prediabetic animals. From our results we conclude that an immunological B-cell destructive process occurs also in long-term normoglycaemic BB rats by participation of mononuclear cells qualitatively not different from those observed in prediabetic animals. Activated T-cells (OX-19+, OX-8+, W3/25+) expressing class II antigens (OX-6+) and the IL-2 receptor (ART-18+) seem to play a significant role in the amplified immunological pancreatic B-cell destruction.

Animals↗
PubMed1990

Phenotyping of lymphocytes following transplantation of allogeneic rat pancreatic islets into streptozotocin-diabetic recipients.

We investigated the phenotypes of peripheral and cervical lymphocytes in allografted pancreatic islet recipients during graft rejection. Grafting 2000 pancreatic islets, obtained from LEW.1A rats, into streptozotocin-diabetic non-immunosuppressed LEW.1W rats acute rejection occurred within 7.0 +/- 0.6 days. Neither the T-cells (W3/13+), nor the T helper (W3/25+) or T-suppressor (OX-8+) lymphocytes were markedly altered during rejection crisis in peripheral or cervical lymphocytes. When using monoclonal antibodies detecting activation markers (OX-17 for class II antigens; ART-18 for interleukin-2 receptor) a significant increase of OX-17+ cells and OX-17+ T-lymphocytes could be observed in lymph node lymphocytes from 24 h after transplantation until 7 days, whereas the peripheral lymphocytes behaved inconspicuously. The results underline the uselessness of peripheral lymphocytes to monitor allogeneic destruction of minimal amounts of grafted tissue by using presently known differentiation and activation markers of lymphocytes.

Animals↗
PubMed1990

Pretreatment with cyclosporine and anti-interleukin 2 receptor antibody abrogates the anti-idiotype response in rat recipients of cardiac allografts.

A 10-day course with ART-18, a mouse monoclonal antibody (mAb) directed against the rat interleukin 2 receptor (IL-2R), prolongs the survival of (LEW x BN)F1 cardiac allografts in LEW recipients to approximately 3 weeks (acute rejection = 8 days, P less than 0.001). We examined host responses against ART-18 idiotype (Id) and mouse immunoglobulin in recipients immunomodulated with ART-18 mAb. Treatment with ART-18 elicited high titers of anti-Id antibodies 14 days after transplantation. However, naive rats given ART-18 before transplantation showed strong anti-Id responses as early as day 4 after engraftment, coinciding with abrogation of the treatment effect (graft survival, approximately 10 days). Preimmunization with irrelevant mouse IgG, which elicited high titers of anti-IgG, did not influence the efficacy of ART-18 upon graft survival (17 days). The use of cyclosporin A (CsA) in conjunction with ART-18 prior to transplantation suppressed the anti-Id response and led to dramatic graft prolongation (greater than 58 days), with two of five grafts surviving indefinitely. This striking effect of CsA plus ART-18 pretreatment did not depend upon CsA per se, as grafts were rejected within 12 days in animals pretreated with CsA alone; in both groups CsA trough levels were comparable. Moreover, administration of CsA before transplantation in concert with control IgG (instead of ART-18) prompted rejection within 2-4 weeks. Thus, discrete interaction(s) between anti-IL-2R mAb and CsA prior to engraftment induces partial host unresponsiveness/tolerance to anti-IL-2R mAb treatment following transplantation and suppresses the neutralizing anti-Id responses, which results in long-term/permanent graft acceptance. This study provides a strategy to overcome the anti-Id response mounted by graft recipients that otherwise limits the efficacy of anti-IL-2R mAb treatment.

Animals↗
PubMed1990

Direct evidence of a functional separation of alloreactive T lymphocytes from bystander cells infiltrating rat allografts. Interleukin 2 receptor-positive cells reacting with the monoclonal antibody ART 18 mediating second-set rejection.

In this study we examined the functional capacity of unseparated, IL-2R positive and IL-2R negative leukocytes infiltrating BN rat hearts or kidneys grafted into allogeneic LEW rats. Upon adoptive transfer into syngeneic LEW recipients, splenocytes or day-3 graft infiltrate cells of either cardiac or renal transplants were ineffective to alter BN cardiac test graft survival (controls 7.8 +/- 0.8 day). However, adoptive transfer of day-5 heart infiltrate cells resulted in a delay of test graft rejection (9.4 +/- 0.7 day, P less than 0.001), while day-5 kidney-graft-infiltrating cells produced second set rejection (6.2 +/- 0.5, P less than 0.001). Specificity controls of day-5 cells infiltrating DA heart or kidney grafts rejected at 7.8 +/- 0.8 or 7.7 +/- 0.5 days. Following separation into IL-2R positive and negative subpopulations by use of the mAB ART 18, IL-2R positive but not IL-2R negative cells caused second set rejection in both the renal and the cardiac model (6.2 +/- 0.4, respectively, 6.3 +/- 0.5 days, P less than 0.001 or P less than 0.005). Furthermore, in the kidney model IL-2R positive nylon-wool nonadherent cells also caused second set rejection (6.2 +/- 0.4, P less than 0.005) suggesting that IL-2R positive T cells present in the graft at maximal infiltration are the mediators of rejection. Thus, it appears that these cells can be phenotypically and functionally separated from bystander cells.

Animals↗
PubMed1990

Evidence that monoclonal antibodies against the 55kD subunit of the rat IL-2 receptor do not inhibit the development of suppressor cells generated in mixed lymphocyte culture.

Although the ability of Ts to prevent allograft rejection has been well established, their intrinsic characteristics and dependence upon lymphokines remain poorly defined. The cells from unmodified LEWxBN bulk 5-day rat MLR inhibit both proliferation in test MLR and generation of CTL, as well as prolonging the survival of donor-specific test cardiac allografts following adoptive transfer. We have examined the effects of a panel of mAb directed against functionally distinct epitopes on the p55 subunit of rat IL-2R on the generation and in vitro/in vivo activity of MLR-generated Ts. ART-18 (which blocks IL-2-dependent T cell growth) was the only mAb from the panel that profoundly suppressed alloreactive T cell proliferation in primary MLR (47.5%). However, the generation of Ts was never affected by any mAb (% suppression in test MLR = 40-60%). Neither ART-18 nor ART-65 (which does not affect T cell proliferation) interfered with the efficacy of Ts to inhibit CTL generation in fresh bulk MLR. Adoptive transfer of cells (3-10 x 10(6] from ART-18 or ART-65-modulated MLR into naive LEW rats prolonged (LEW x BN)F1 test cardiac allograft survival to 11-13 days (P less than 0.05 as compared with acutely rejecting hosts). All in vitro and in vivo effects exerted by MLR-generated cells were antigen-specific. In unmodified MLR, Ts were IL-2R+ (ca. 50% of total blasts), as shown by cell separation using magnetic beads. In contrast, in MLR with ART-18 added, Ts were primarily IL-2R- (ca. 10% of blasts). Thus, antirat p55 subunit IL-2R mAb do not inhibit MLR-generated Ts functionally operative in vitro and in vivo. IL-2R- Ts precursors requiring lymphokine(s) other than IL-2 may differentiate into IL-2-dependent Ts effectors. Such divergent IL-2 requirements for Ts growth in vitro may explain the Ts-sparing effects in allograft recipients treated with anti-IL-2R mAb.

Animals↗