Murine Thymic Cells Research Articles

Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice.

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear’s vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living.

Wnt4 signaling is associated with the decrease of proliferation and increase of apoptosis during age-related thymic involution.

The thymus is a central lymphoid organ that is responsible for T-lymphocyte development and maturation. Through negative and positive selection, lymphoid progenitor cells, which initiate from the bone marrow, develop into mature T cells in the thymus, and are subsequently involved in peripheral cell immunity. It has been reported that the Wnt signaling pathway exists widely in thymic epithelial cells and T lymphocytes. Wnt signaling affects the shape and function of thymic epithelial cells and has an important role in maintaining pro‑T‑cells, and in the subsequent T‑cell differentiation. Previous studies have demonstrated that the Wnt signaling pathway participates in age‑associated thymic involution. In the present study alterations in proliferation and apoptosis were investigated in murine thymic cells during aging. The results of the present study demonstrated that the aged thymus was characterized by markedly decreased cell numbers, as well as decreased proliferation and increased apoptosis. Concurrently, age‑associated changes in thymic cell number and function were accompanied by a decrease in the transcription levels of Wnt4, and downregulation of forkhead box N1 and B‑cell lymphoma‑extra large, which are two target genes of the Wnt4 signaling pathway. In vitro studies demonstrated that activation of the Wnt4 signaling pathway promotes mouse thymus epithelial cell 1 (MTEC1) cell proliferation, and that Wnt4 signaling modulation alleviates dexamethasone‑mediated MTEC1 cell apoptosis. These results suggest that normal expression levels of Wnt4 have a critical role in maintaining the balance between cell proliferation and apoptosis. Alterations in the Wnt signaling pathway may disrupt the epithelial network structure of the thymus, eventually leading to microenvironmental damage. Therefore, further studies regarding the effects of the Wnt signaling pathway on thymus development and age-related thymic involution, may be beneficial for improving the health conditions of the elderly.

Proliferative Activity and Subpopulation Pattern of Cells in Murine Thymus and Spleen on the Model of Graft-Versus-Host Chronic Reaction under Various Diurnal Regimens of IL-2 Administration

We studied the effects of IL-2 administration at different times of day to female B6D2F1 mice with experimental immunopathology, graft-versus-host chronic reaction induced by the administration of lymphoid cells from parental female DBA/2 mice. Recombinant murine IL-2 was injected subcutaneously at 10.00 (group 1) or 16.00 (group 2) after the first cell transfer. Evening administration of IL-2 in contrast to its morning injection prevented cell proliferation in the thymus and spleen, decreased the number of apoptotic splenocytes, significantly stimulated differentiation processes in the thymus, increased the amount of CD4 (+) 25 (high) thymocytes, and decreased the number of CD4 (+) 27 (low) splenocytes. These findings can serve as a prerequisite for the development of chronotherapeutic schemes for immunocorrection.

74 : IL-7R: Mutations in T-ALL and polymorphisms in autoimmunity

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from leukemic transformation of T-cell progenitors in the thymus. It accounts for approximately 15% of ALL cases in childhood and 20–25% in adults and is a leading cause of death in children. IL-7 and its receptor (IL-7R) play a critical role in normal T-cell development and homeostasis. Mutations in IL-7R were identified in 9% of pediatric T-ALL patients. These mutations usually involved insertions of three amino acids including cysteine and proline in the extracellular juxtamembrane region. WT or mutant forms of the human IL-7R (hIL-7R) from patients were retrovirally transfected into an IL-7-dependent murine thymic cell line D1. Mutant hIL-7Rs induced ligand-independent activation of the Jak-Stat and PI3K pathways, cell survival and proliferation. Notably, mutant hIL-7R-expressing D1 cells formed subcutaneous tumors in Rag1-/- mice, with substantial infiltration into various organs that are normally affected in advanced stages of T-ALL, such as bone marrow, liver, lymph nodes and spleen. Further functional assays revealed that mutant hIL-7Rs constitutive signaling required homodimerization via cysteines in the inserted sequences and downstream Jak1 activation, and was IL-7, γ c and Jak3-independent. Jak inhibitors were effective in blocking proliferation and survival of transformed cells. The hotspot for insertions lies in exon 6 in precisely the same region as a coding polymorphism regulating risk for MS and other autoimmune diseases, and we observe that this polymorphism affects strength of signaling. Our findings indicate that IL-7R mutations drive T-ALL, whereas polymorphisms that increase signaling promote autoimmunity, implicating IL-7R and Jak1 as therapeutic targets in these diseases.

O023 Oncogenic IL-7R gain-of-function mutations in childhood T-ALL

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy resulting from leukemic transformation of T-cell progenitors in the thymus. It accounts for approximately 15% of ALL cases in childhood and 20–25% in adults and is a leading cause of death in children. IL-7 and its receptor (IL-7R) play a critical role in normal T-cell development and homeostasis. Methods The IL-7R gene was sequenced in T-ALL from three cohorts. Results Mutations in IL-7R were identified in 9% of pediatric T-ALL patients. These mutations usually involved insertions of three amino acids including cysteine and proline in the extracellular juxtamembrane region. WT or mutant forms of the human IL-7R (hIL-7R) from patients were retrovirally transfected into an IL-7-dependent murine thymic cell line D1. Mutant hIL-7Rs induced ligand-independent activation of the Jak-Stat and PI3K pathways, cell survival and proliferation. Notably, mutant hIL-7R-expressing D1 cells induced subcutaneous tumors in Rag1-/- mice, with substantial infiltration into various organs that are normally affected in advanced stages of T-ALL, such as bone marrow, liver, lymph nodes and spleen. Further functional assays revealed that mutant hIL-7Rs constitutive signaling required homodimerization via cysteines in the inserted sequences and downstream Jak1 activation, and was IL-7, gc and Jak3-independent. Conclusion Our findings indicate that hIL-7R mutational activation drives T-ALL leukemogenesis and implicate IL-7R and Jak1 as therapeutic targets in T-ALL.

GM-CSF induces podoplanin expression during dendritic cell differentiation (111.56)

Abstract Podoplanin is a mucin-type transmembrane glycoprotein containing extensive Oglycosylation in extracellular domain and two phosphorylation sites in a short cytoplasmic tail. It is expressed in kidney glomerular epithelial cells, lymphatic endothelium and subcapsular epithelial cells in murine thymus. Recently, podoplanin was found on murine F4/80 positive peritoneal macrophages and human follicular dendritic cells (FDCs) indicating that podoplanin is involved in immune response. We have reported that podoplanin is induced during Th9 and Th17 polarization. Here, we studied podoplanin expression on different types of murine myeloid cells. We found that podoplanin was not detectable on circulating and bone marrow derived neutrophils, monocytes, basophils and esinophils, its expression level on neutrophil and monocytes was not inducible followed by fMLP or TNF-a stimulation. Podoplanin was also not detectable on spleen and lymph node resided CD11b+macrophages, CD11c+ dendritic cells (cDCs) and CD8+ plasmacytoid dendritic cells (pDCs). However, podoplanin expression level has been dramatically increased on GM-CSF driven bone marrow-derived dendritic cells. These data suggest that podoplanin can be induced during GM-CSF driven dendritic cell differentiation, indicating that podoplanin may be a transient marker during dendritic cells differentiation and maturation. The mechanism of GM-CSF induced podoplanin and it significance need to be further investigated.

Screening for novel lead compounds increasing insulin expression in medullary thymic epithelial cells

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance to pancreatic beta-cells. Thymic insulin expression modulation might be an important drug target for preventing type 1 diabetes. We performed a high-throughput screening to identify compounds with such activity. A reporter plasmid was constructed with the human insulin promoter sequence including a short allele of the upstream variable number tandem repeat (VNTR) sequence (32 repeats), subcloned into the pGL4.17 vector. The plasmid was stably transfected into an insulin-transcribing medullary thymic epithelial cell (mTEC) line. Primary high-throughput screening assays were carried out by stimulating with candidate compounds for 24h, and the activity of luciferase was measured. Positive compounds were further validated by real-time PCR. Of 19,707 compounds, we identified one compound that could enhance mTEC insulin expression, as confirmed by real-time PCR. We also observed that transfection with the autoimmune regulator (AIRE) increased endogenous AIRE expression in mTECs. Our insulin-VNTRI-promoter reporter system is consistent with the insulin expression regulation in mTECs, and one compound that was identified could increase insulin expression in mTECs. A positive feedback effect of AIRE in mTECS was observed. Whether these efforts in murine thymus cells apply to humans remains to be determined.

Continuous Activation of the CD122/STAT-5 Signaling Pathway during Selection of Antigen-Specific Regulatory T Cells in the Murine Thymus

Signaling events affecting thymic selection of un-manipulated polyclonal natural CD25+foxp3+ regulatory T cells (nTreg) have not been established ex vivo. Here, we report a higher frequency of phosphorylated STAT-5 (pSTAT-5) in nTreg cells in the adult murine thymus and to a lesser extent in the periphery, compared to other CD4+CD8− subsets. In the neonatal thymus, the numbers of pSTAT-5+ cells in CD25+foxp3− and nTreg cells increased in parallel, suggesting that pSTAT-5+CD25+foxp3− cells might represent the precursors of foxp3+ regulatory T cells. This “specific” pSTAT-5 expression detected in nTreg cells ex vivo was likely due to a very recent signal given by IL-2/IL-15 cytokines in vivo since (i) it disappeared rapidly if cells were left unstimulated in vitro and (ii) was also observed if total thymocytes were stimulated in vitro with saturating amounts of IL-2 and/or IL-15 but not IL-7. Interestingly, STAT-5 activation upon IL-2 stimulation correlated better with foxp3 and CD122 than with CD25 expression. Finally, we show that expression of an endogenous superantigen strongly affected the early Treg cell repertoire but not the proportion of pSTAT-5+ cells within this repertoire. Our results reveal that continuous activation of the CD122/STAT-5 signaling pathway characterize regulatory lineage differentiation in the murine thymus.

Podoplanin is involved in Th17 cell polarization (113.17)

Abstract Podoplanin is a mucin-type transmembrane glycoprotein containing extensive O-glycosylation in extracellular domain and two phosphorylation sites in a short cytoplasmic tail. It is expressed in kidney glomerular epithelial cells, lymphatic endothelium and subcapsular epithelial cells in murine thymus. Recently, podoplanin was found on F4/80 positive peritoneal macrophages, indicating that podoplanin is involved in immune response. However, the physiologic function of podoplanin is still unknown. To study whether podoplanin is involved in adaptive immunity, we measured its expression on lymphocyts. The data showed that podoplanin was not detectable on freshly isolated T and B lymphocytes from peripheral blood, spleen and thymus, its expression is also non-inducible after stimulation with PMA, Concanavalin A or LPS. Surprisingly, we found that the podoplanin expression on CD4+ cells was dramatically increased after 3 days culture with Th17 polarization medium compose of immobilized CD3/CD28, IL-6, IL-4, TGFb, IFN-r and IL-23, which showed more than 60% cells were podoplanin positive. However, its expression is not able to be induced on Th1 and Th2 polarized cells. By detecting IL-17 production, we interestingly found that the IL-17+ cells can be divided into podplaninhigh and podoplaninlow populations. The significance of the differential expression on Th17 cells needs to be further investigated.

Interleukin (IL)‐7 promotes T‐cell survival by stabilization of Mcl‐1

IL‐7 is required for T‐cell development both in mouse and humans and for T‐cell homeostasis in the periphery. Some of the downstream signal transducers from the IL‐7 receptor are members of the Bcl‐2 family of proteins associated with mitochondrial apoptosis. Mcl‐1, one of the anti‐apoptotic members of Bcl‐2 family, became of particular interest when it was discovered that IL‐7 upregulates Mcl‐1 mRNA expression in T cells. This suggests that Mcl‐1 may contribute to the survival functions of IL‐7. In this study, we showed that the deficiency of T–cell development in IL‐7Rα KO mice could be rescued by reconstitution of IL‐7Rα deficient bone marrow by retrovirus expressing a mouse Mcl‐1 construct. To further investigate the molecular mechanism of IL‐7 regulation of Mcl‐1, we used an IL‐7‐dependent murine thymic cell line D1. Deprivation of IL‐7 from D1 cells or lymph node cells caused Mcl‐1 protein degradation by 6 hrs. Overexpression of Mcl‐1 protected D1 cells from the death induced by IL‐7 deprivation. Compared to intact Mcl‐1, the truncated form of Mcl‐1 (Δ375), with deletion of 375 nucleotides from its 5′end, showed more potent anti‐apoptotic activity, suggesting the 5′end of Mcl‐1 might contain regulatory domains. In human Mcl‐1 the lysine in the 5′ end of the gene was identified as the target site for ubiquitin. Based on this finding, we hypothesized that the lysine found in the mouse Mcl‐1 would share this function. To test this hypothesis, we mutated the first three lysine residues (39, 80, and 117) to alanine at mouse Mcl‐1 (Mcl 3KR) and tested its protein stability and its ability to prevent apoptosis. Mcl 3KR showed enhanced protein stability, and also provided D1 cells with more protection, compared to WT Mcl‐1, after 3 to 4 days of IL‐7 deprivation. Our findings suggest that Mcl‐1 plays an important role protection of T‐cell survival by IL‐7. IL‐7 strongly promotes Mcl‐1 stability, possible by controlling lysine‐directed ubiquitination.

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A (1–17) as a non-substrate inhibitor

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V max = 39 μmol/min/10 6 cells, vs 3.7 μmol/min/10 6 cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A (1–17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A (1–17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A (1–17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A (1–17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A (1–17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A (1–17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.

Murine thymic plasmacytoid dendritic cells.

We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c(+)B220(+) subset expresses Ly6C(high) and MHC class II(low) in contrast with previously described thymic DC (CD11c(+)B220(-) cells). Freshly isolated thymic CD11c(+)B220(+) cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN-alpha following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL-12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN-producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.

Macrophages are involved in DNA degradation of apoptotic cells in murine thymus after administration of hydrocortisone

In the present study, we undertook kinetic analyses of DNA degradation and acid DNase activity in murine thymus after administration of hydrocortisone. Hydrocortisone induced apoptosis in thymocytes, and a large number of cortical thymocytes became TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling)-positive (TUNEL+). F4/80+ macrophages infiltrated through the cortico-medullay junction into the cortical region, and thereafter engulfed apoptotic cells in the cortex of thymus. The distribution of acid DNase-active cells appeared to be similar to that of F4/80+ macrophages. Eighteen hours after the injection, although the foci of apoptotic cells were situated within massively distended F4/80+ macrophages, oligonucleosomal DNA fragments on an agarose gel were undetectable. Our results showed that macrophages were involved in the disappearance of oligonucleosomal DNA fragments in apoptotic thymocytes. Taken together, macrophages play a role in the hydrolysis of DNA in apoptotic cells upon their phagocytosis of the dead cells.

Macrophages are involved in DNA degradation of apoptotic cells in murine thymus after administration of hydrocortisone.

In the present study, we undertook kinetic analyses of DNA degradation and acid DNase activity in murine thymus after administration of hydrocortisone. Hydrocortisone induced apoptosis in thymocytes, and a large number of cortical thymocytes became TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling)-positive (TUNEL+). F4/80+ macrophages infiltrated through the cortico-medullay junction into the cortical region, and thereafter engulfed apoptotic cells in the cortex of thymus. The distribution of acid DNase-active cells appeared to be similar to that of F4/80+ macrophages. Eighteen hours after the injection, although the foci of apoptotic cells were situated within massively distended F4/80+ macrophages, oligonucleosomal DNA fragments on an agarose gel were undetectable. Our results showed that macrophages were involved in the disappearance of oligonucleosomal DNA fragments in apoptotic thymocytes. Taken together, macrophages play a role in the hydrolysis of DNA in apoptotic cells upon their phagocytosis of the dead cells.

Enhanced Complement Sensitivity of NK-T Cells in Murine Thymus and Spleen Associated with Presence of Serum Immunoglobulin

In vitro treatment of thymocytes and splenocytes with rabbit complement (C') alone induced significant reductions in the proportion of NK-T cells in murine system. The reduction appeared to be prominent in the thymic NK-T cells compared to that in splenic NK-T cells. No reductions were detected in other populations, such as T, B and NK cells. Thus, NK-T cells lineage-specifically showed the enhanced C' sensitivity. However, NK-T cells in T cell receptor (TCR) transgenic mice of RAG-/- background that lack B cells and antibodies exhibited no C' sensitivity. On the other hand those from the same TCR transgenic mice of RAG intact background that have a normal population of B cells and antibodies showed the C' sensitivity similar to that in normal mice. These findings suggest that the enhanced C' sensitivity observed in the NK-T cell population is associated with the NK-T specific autoantibodies. Indeed, we found that a subset of NK-T cells in the thymus bound mouse immunoglobulins. Similar observations were obtained with several strains of lupus model mice, some of which show a decrease of NK-T cells with aging. Possible roles of the enhanced C' sensitivity of NK-T cells in pathophysiological conditions in various mouse strains including lupus models are discussed.

Analysis of fluorescence lifetime and quenching of FITC-conjugated antibodies on cells by phase-sensitive flow cytometry.

Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another results in self-quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity antibodies relative to one another on a highly labeled cell surface. Since self-quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase-sensitive detection method to investigate the impact of self-quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti-mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self-quenching effects expected at high densities of FITC molecules.

Cholinergic Stimulation Modulates Apoptosis and Differentiation of Murine Thymocytes via a Nicotinic Effect on Thymic Epithelium

Effects of cholinergic agonists/antagonists on apoptosis and differentiation of murine thymus cells were investigated in two in vitro models. Treatment with 10−7 M carbachol was found to counteract the effects of a cortical thymus epithelial cell line (TEC 1.4), on apoptosis and the ratio of CD4 CD8 DP/DN cells in cocultured fetal tjymus lobes. A medullary line (TEC 2.3) did not influence apoptosis in fetal thymus lobes. Both TEC lines had the same strong apoptotic effect on thymus cells in suspension, but only the effect of TEC 1.4 was counteracted by carbachol. This cholinergic influence on TEC 1.4 cells is mediated via nicotinic cholinergic receptors, since d-tubocurarine, but not atropine, effectively blocked the effect of carbachol. The results suggest that cholinergic signals to thymic epithelial cells may have regulatory influence on thymic differentiation and selection processes.

A reliable method for quantitating chromatin fragments by flow cytometry to predict the effect of total body irradiation and hyperthermia on mice

The frequencies of chromatin fragments, including micronuclei, in murine thymus cells, spleen cells and bone marrow cells have been used as a quantitative indicator of gamma-ray induced chromosome damage and could be used to screen potential radioprotective agents as well. The yield of chromatin fragments induced in mice receiving different dosage levels of total body irradiation alone and in mice also given whole body hyperthermia as a potent radioprotector were assessed by flow cytometric analysis. Our results demonstrated that chromatin fragments induced by irradiation in vivo was clearly dose-dependent and that chromatin fragments could potentially serve as a biological indicator of radiation damage. One hour of whole body hyperthermia at 40°C (±0.2°C) given 20 hours before a lethal dosage (900 cGy) of total body irradiation protects 100% of DBA/2 mice from an LD 100/16 irradiation dose (dose of irradiation that killed 100% of the mice in 16 days). This is in good agreement with the percent of chromatin fragments formed in the cells of the protected animals, which showed no significant difference from those observed in the normal mice. The results indicate that whole body hyperthermia protected the thymus and bone marrow from irradiation damage. This study provides further evidence which supports that whole body hyperthermia can act as a potent radioprotector in vivo. Measurement of the frequencies of chromatin fragments by flow cytometry is simple and reliable. The method can be applied to screen radioprotective agents.

The immunosuppressive effect of acute doses of emetine on murine thymic cells

Emetine — a general inhibitor of protein synthesis — was investigated for its ability to depress specific immune response in animal models. A single dose (33 mg/kg) of emetine administered subcutaneously to mice markedly decreased thymus weight and thymic cell numbers. DNA, RNA and protein synthesis of thymic cells were reduced by 90, 50 and 65%, respectively. RNA synthesis was the first process to recover followed by the reconstitution of DNA and protein synthesizing capacity. Histological evidence revealed an effect on the cortical region of thymus upon emetine administration. Results suggest that emetine exerts an immunosuppressive effect on T-cell maturation in the thymus. These findings may enhance a therapeutic interest in the drug.

Identification of c-myb (chicken), c-myb (mouse) and v-myb (AMV) protein products by immunoprecipitation with antibodies directed against a synthetic peptide

A synthetic nonadecapeptide (IL 19) derived from a sequence of v-myb was covalently bound to haemocyanin and used for immunization. Anti-IL 19 serum immunoprecipitated a 75 kDa protein in the lysate of metabolically labelled chicken and murine thymus cells. Presaturation of the serum with IL 19 abolished this immunoprecipitation, thus indicating that the product of c-myb in both chicken and murine thymuses is the 75 kDa protein (p75 c-myb). Anti IL 19 serum also precipitated p48 c-myb) in the lysate of nonproducer myeloblasts.