Armenian Hamster Research Articles

Host species-specific activity of the poxvirus PKR inhibitors E3 and K3 mediate host range function.

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. The expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix αG was resistant to wild-type VACV infection and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA.IMPORTANCEThe molecular mechanisms that govern the host range of viruses are incompletely understood. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. The current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters double-stranded (ds) RNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete and that dsRNA sequestration is not the primary mechanism for E3 activity.

First reported case of a histiocytic sarcoma in an Armenian hamster (Cricetulus migratorius).

A 14-month-old male Armenian hamster (Cricetulus migratorius) presented with a spontaneous, subcutaneous, firm mass (4.0 × 2.0 × 1.5 cm) on the ventral neck extending towards the cheek pouch causing multifocal small oral ulcerations. This animal was immunized subcutaneously on the dorsal neck for the development of monoclonal antibodies seven months before presentation. The animal was euthanized and necropsy was performed. Histopathology of the mass showed a well demarcated, multilobulated, unencapsulated, highly cellular, neoplastic mass composed of spindle cells arranged in interlacing streams and bundles, with a moderate amount of fibrovascular stroma. The neoplastic cells exhibited indistinct cell borders and a moderate to large amount of eosinophilic, fibrillar cytoplasm, marked anisocytosis and anisokaryosis, binucleated and multinucleated cells, and high mitotic rate. Based on the histomorphologic features of the mass, and the presence of renal tubular hyaline globules and myeloid hyperplasia in the bone marrow, a diagnosis of histiocytic sarcoma was made. The presumptive diagnosis was confirmed by immunohistochemistry, upon which the neoplastic cells showed strong immunoreactivity for the histiocytic cell markers Iba1 and CD11b. Histiocytic sarcomas have been reported in Syrian (Mesocricetus auratus) and Siberian dwarf (Phodopus sungorus) hamsters but, to our knowledge, the current report represents the first case of histiocytic sarcoma described in an Armenian hamster. It is plausible to consider the animal's experimental immunization history and the development of the histiocytic sarcoma to be related. An association between adjuvanted vaccines and soft-tissue sarcomas has been described in cats and referred to as feline injection-site sarcomas.

Effects of skin γδ T lymphocytes on wound healing of mice through regulating proliferation and differentiation of mice epidermal cells

Effects of skin γδ T lymphocytes on wound healing of mice through regulating proliferation and differentiation of mice epidermal cells

Agonistic Anti-TIGIT Treatment Inhibits T Cell Responses in LDLr Deficient Mice without Affecting Atherosclerotic Lesion Development

ObjectiveCo-stimulatory and co-inhibitory molecules are mainly expressed on T cells and antigen presenting cells and strongly orchestrate adaptive immune responses. Whereas co-stimulatory molecules enhance immune responses, signaling via co-inhibitory molecules dampens the immune system, thereby showing great therapeutic potential to prevent cardiovascular diseases. Signaling via co-inhibitory T cell immunoglobulin and ITIM domain (TIGIT) directly inhibits T cell activation and proliferation, and therefore represents a novel therapeutic candidate to specifically dampen pro-atherogenic T cell reactivity. In the present study, we used an agonistic anti-TIGIT antibody to determine the effect of excessive TIGIT-signaling on atherosclerosis.Methods and ResultsTIGIT was upregulated on CD4+ T cells isolated from mice fed a Western-type diet in comparison with mice fed a chow diet. Agonistic anti-TIGIT suppressed T cell activation and proliferation both in vitro and in vivo. However, agonistic anti-TIGIT treatment of LDLr−/− mice fed a Western-type diet for 4 or 8 weeks did not affect atherosclerotic lesion development in comparison with PBS and Armenian Hamster IgG treatment. Furthermore, elevated percentages of dendritic cells were observed in the blood and spleen of agonistic anti-TIGIT-treated mice. Additionally, these cells showed an increased activation status but decreased IL-10 production.ConclusionsDespite the inhibition of splenic T cell responses, agonistic anti-TIGIT treatment does not affect initial atherosclerosis development, possibly due to increased activity of dendritic cells.

Matrix Metalloproteinase-12 Deficiency Worsens Relapsing-Remitting Experimental Autoimmune Encephalomyelitis in Association with Cytokine and Chemokine Dysregulation

The elevation of several members of the matrix metalloproteinase (MMP) family promotes the pathophysiology of both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Nonetheless, given the multiple activities of MMPs, it remains possible that increased levels of a particular MMP may have beneficial functions during disease progression. We reported previously that MMP-12(-/-) mice of the 129/SvEv strain had a poorer EAE outcome than wild-type controls. However, we did not determine further differences in disease profiles between these groups. Using the EAE model in 129/SvEv mice, we report that disease in both wild-type and MMP-12(-/-) mice follows a relapsing-remitting course. Although both mouse groups had similar clinical onsets, subsequent relapses were more severe in MMP-12(-/-) mice; their residual disability at remission was also higher compared with wild-type controls. The worsened relapses and remissions in MMP-12(-/-) mice occurred despite a deficiency of the antigen recall capacity of lymph node-derived cells as well as a reduction in the proportion of macrophages in the spinal cord during the chronic phase of EAE. Significantly, large increases of levels of chemokines and cytokines were found in the spinal cords of MMP-12(-/-) mice during chronic EAE. These results highlight MMP-12 as a beneficial enzyme in EAE and suggest that therapeutic interventions in multiple sclerosis should avoid targeting MMP-12.

Generation and characterization of a novel anti-rat CD40L antibody with inhibitory activities in vitro and in vivo

The CD40–CD40L interaction plays a critical role in cell-mediated immune responses. Blocking this interaction has been shown to be beneficial in the treatment of various diseases studied in murine models. Although rats are widely used to test therapeutic strategies in several disease models, a monoclonal antibody (mAb) to block the CD40–CD40L interaction in rats is not broadly available. In the present study we generated Armenian hamster fibroblasts expressing rat CD40L and used these to generate a novel anti-rat CD40L mAb (AS1). In vitro studies showed that AS1 was able to block CD40L-induced DC maturation and B cell proliferation. Most importantly, in vivo, AS1 inhibited B cell responses in a dose-dependent fashion, as measured by the production of OVA specific antibodies after subcutaneous immunization with OVA. AS1 was shown to be a powerful tool to modulate Ag presentation in vitro and in vivo. Elucidating the effect of AS1 in various rat models for human diseases will provide more insight into blocking the CD40–CD40L interaction as a therapeutic strategy to prevent human diseases.

PGAP1 Knock-out Mice Show Otocephaly and Male Infertility

A palmitate linked to the inositol in glycosylphosphatidylinositol (GPI) is removed in the endoplasmic reticulum immediately after the conjugation of GPI with proteins in most cells. Previously, we identified PGAP1 (post GPI attachment to proteins 1) as a GPI inositoldeacylase that removes the palmitate from inositol. A defect in PGAP1 caused a delay in the transport of GPI-anchored proteins (GPI-APs) from the endoplasmic reticulum to the cell surface in Chinese hamster ovary cells, although the cell-surface expression of GPI-APs in the steady state was normal. Nevertheless, in most cells, GPI-APs undergo deacylation. To elucidate the biological significance of PGAP1 in vivo, we established PGAP1 knock-out mice. Most PGAP1 knock-out mice showed otocephaly, a developmental defect, and died right after birth. However, some survived with growth retardation. Male knock-out mice showed severely reduced fertility despite the capability of ejaculation. Their spermatozoa were normal in number, motility, and ability to ascend the uterus, but were unable to go into the oviduct. In vitro, PGAP1-deficient spermatozoa showed weak attachment to the zona pellucida and a severely diminished rate of fertilization. Therefore, an extra acyl chain in GPI anchors caused severe deleterious effects to development and sperm function.

The human EGF-TM7 receptor EMR3 is a marker for mature granulocytes

EMR3 is a member of the epidermal growth factor-seven-transmembrane (EGF-TM7) family of adhesion class TM7 receptors. This family also comprises CD97, EMR1, EMR2, and EMR4. To characterize human EMR3 at the protein level, we generated Armenian hamster mAb. Using the mAb 3D7, we here demonstrate that EMR3, like other EGF-TM7 receptors, is expressed at the cell surface as a heterodimeric molecule consisting of a long extracellular alpha-chain, which possesses at its N-terminus EGF-like domains and a membrane-spanning beta-chain. Flow cytometric analysis revealed that all types of myeloid cells express EMR3. In peripheral blood, the highest expression of EMR3 was found on granulocytes. More mature CD16(+) monocytes express high levels of EMR3, and CD16(-) monocytes and myeloid dendritic cells (DC) are EMR3(dim/low). Lymphocytes and plasmacytoid DC are EMR3(-). It is interesting that in contrast with CD97 and EMR2, CD34(+)CD33(-)/CD38(-) committed hematopoietic stem cells and CD34(+)CD33(+)/CD38(+) progenitors in bone marrow do not express EMR3. In vitro differentiation of HL-60 cells and CD34(+) progenitor cells revealed that EMR3 is only up-regulated during late granulopoiesis. These results demonstrate that the expression of EGF-TM7 receptors on myeloid cells is differentially regulated. EMR3 is the first family member found mainly on granulocytes.

Differential Requirement for RhoA GTPase Depending on the Cellular Localization of Protein Kinase D

This study explores the links between the GTPase RhoA and the serine kinase protein kinase D (PKD) during thymocyte development. The rationale is that RhoA and PKD regulate common biological responses during T cell development, but there is nothing known about their interdependence. In fibroblasts, Rho function is required for activation of PKD catalytic activity. However, the data show that activation of Rho is neither sufficient nor essential for PKD activation in T cells. One alternative explanation for the apparent convergence of PKD and Rho signaling in T cells is that PKD responses might be Rho-dependent. To address this latter possibility, we probed the Rho requirements for the actions of constitutively active PKD mutants in pre-T cells of transgenic mice. Active PKD can localize to either the plasma membrane or the cytosol, and we therefore compared the Rho requirements for the actions of membrane- or cytosol-localized PKD. Here we show that membrane-localized PKD regulation of pre-T cell differentiation is Rho-dependent, but the actions of cytosol-localized PKD are not. These studies demonstrate that a Rho requirement for PKD activation is not ubiquitous. Moreover, links between PKD and Rho are determined by the cellular location of PKD.

Activation of Wnt signaling in the intestinal mucosa of Apc+/min mice does not cause overexpression of the receptor tyrosine kinase Met

The receptor tyrosine kinase MET is overexpressed in human colorectal adenomas and carcinomas, suggesting an instrumental role for MET signaling in the onset and progression of colorectal cancer. To corroborate this role, animal models are needed. To study the expression of Met in the normal and neoplastic mouse intestine, we generated an Armenian hamster monoclonal antibody against mouse Met. By using this antibody in immunohistochemical studies, we observed strong Met expression in fetal mouse intestinal epithelial cells. In contrast, in the intestines of adult mice, Met expression was very low whereas the protein was undetectable on the neoplastic epithelium of intestinal adenomas in Apc+/min mice. By immunoblotting, we were also unable to detect Met in intestinal adenomas, whereas Met mRNA levels in microdissected adenomas were very low. The absence of detectable Met protein expression in adenomas of Apc+/min mice contrasts sharply with the vast overexpression of the protein in adenomas of humans with familial adenomatous polyposis or sporadic colorectal carcinomas. Our results imply that deregulation of Wnt signaling in mouse--unlike in human--intestinal epithelium does not result in Met overexpression. Our findings thus reveal important interspecies differences in the regulation of Met expression during intestinal tumorigenesis.

Dissecting linear and conformational epitopes on the native thyrotropin receptor.

The TSH receptor (TSHR) is the primary antigen in Graves' disease. In this condition, autoantibodies to the TSHR that have intrinsic thyroid-stimulating activity develop. We studied the epitopes on the native TSHR using polyclonal antisera and monoclonal antibodies (mAbs) derived from an Armenian hamster model of Graves' disease. Of 14 hamster mAbs analyzed, five were shown to bind to conformational epitopes including one mAb with potent thyroid-stimulating activity. Overlapping conformational epitopes were determined by cell-binding competition assays using fluorescently labeled mAbs. We identified two distinct conformational epitopes: epitope A for both stimulating and blocking mAbs and epitope B for only blocking mAbs. Examination of an additional three mouse-derived stimulating TSHR-mAbs also showed exclusive binding to epitope A. The remaining nine hamster-derived mAbs were neutral or low-affinity blocking antibodies that recognized linear epitopes within the TSHR cleaved region (residues 316-366) (epitope C). Serum from the immunized hamsters also recognized conformational epitopes A and B but, in addition, also contained high levels of TSHR-Abs interacting within the linear epitope C region. In summary, these studies indicated that the natively conformed TSHR had a restricted set of epitopes recognized by TSHR-mAbs and that the binding site for stimulating TSHR-Abs was highly conserved. However, high-affinity TSHR-blocking antibodies recognized two conformational epitopes, one of which was indistinguishable from the thyroid-stimulating epitope. Hence, TSHR-stimulating and blocking antibodies cannot be distinguished purely on the basis of their conformational epitope recognition.

Preparation of Monoclonal Antibodies to Murine Platelet Glycoprotein IIb/IIIa (αIIbβ3) and Other Proteins from Hamster–Mouse Interspecies Hybridomas

To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from β3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet–fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet–fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.

Estrogen induces cytokeratin aggregation in primary cultures of Armenian hamster hepatocytes

The effect of estrogen administration to cultured Armenian hamster was studied. Isolated Armenian hamster hepatocytes were cultured in RPMI medium supplemented with β-estradiol (E2). β-estradiol treatment for 24–48 hr induced cytoplasmic inclusion bodies which by immunocytochemistry were positive for cytokeratin (CK) 8, CK 18, and ubiquitin but negative for CK 7 and CK 19. These inclusion bodies appeared as filamentous tangles or amorphous aggregates when observed by electron microscopy. F-actin, tubulin, and desmosomes were not influenced by the presence of the inclusion bodies. Addition of ethanol to culture medium increased the incidence of the inclusion formation. In combination with 0.5% ethanol 1 μM of E2 induced five to six times more inclusion bodies, while the number of inclusion bodies decreased when epidermal growth factor (EGF) was added to the medium in combination with E2. This reduction effect was nullified by treatment with anti-EGF receptor antibody. These findings suggest that E2 treatment to Armenian hamster hepatocytes in vitro induces Mallory body-like inclusions whose incidence can be influenced by addition of ethanol or EGF to the culture medium. Cell Motil. Cytoskeleton 43:35–42, 1999. © 1999 Wiley-Liss, Inc.

Estrogen induces cytokeratin aggregation in primary cultures of Armenian hamster hepatocytes

The effect of estrogen administration to cultured Armenian hamster was studied. Isolated Armenian hamster hepatocytes were cultured in RPMI medium supplemented with beta-estradiol (E2). Beta-estradiol treatment for 24-48 hr induced cytoplasmic inclusion bodies which by immunocytochemistry were positive for cytokeratin (CK) 8, CK 18, and ubiquitin but negative for CK 7 and CK 19. These inclusion bodies appeared as filamentous tangles or amorphous aggregates when observed by electron microscopy. F-actin, tubulin, and desmosomes were not influenced by the presence of the inclusion bodies. Addition of ethanol to culture medium increased the incidence of the inclusion formation. In combination with 0.5% ethanol 1 microM of E2 induced five to six times more inclusion bodies, while the number of inclusion bodies decreased when epidermal growth factor (EGF) was added to the medium in combination with E2. This reduction effect was nullified by treatment with anti-EGF receptor antibody. These findings suggest that E2 treatment to Armenian hamster hepatocytes in vitro induces Mallory body-like inclusions whose incidence can be influenced by addition of ethanol or EGF to the culture medium.

Comparison of the mRNA sequences for Pi class glutathione transferases in different hamster species and the corresponding enzyme activities with anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide

Glutathione S-transferase (GST) of class Pi (GST Pi) is known to detoxify the mutagenic and carcinogenic (+)-anti-benzo[a]pyrene-7, 8-dihydrodiol 9,10-epoxide [(+)-anti-BPDE] by conjugation with glutathione. Previously, we have shown that Chinese hamster V79 cells contain GST Pi, but seem to lack the capacity to conjugate (+)-anti-BPDE, although these cells do conjugate other substrates with GSH [Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701-1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719-1723; Swedmark and Jenssen (1994) Gene 139, 251-256]. In the present study we have compared four cell lines derived from different hamster species with respect to GST cDNA sequences and capacity to conjugate (+)-or(-)-anti-BPDE. The cell lines were V79 and Chinese hamster ovary cells (CHO), Armenian hamster lung (AHL) cells and baby hamster kidney (BHK) cells. The sequencing revealed a complete homology between the V79 and CHO cDNA for GST Pi, whereas the corresponding amino acid sequences predicted from the corresponding AHL and BHK cDNAs differed by six and nine amino acids, respectively, from the predicted V79 sequence. None of these changes alone was found to influence the xenobiotic substrate-binding site. The cytosolic fractions from BHK and AHL cells were found to catalyse conjugation of (+)-anti-BPDE with GSH, whereas the corresponding activity in CHO cells was non-detectable. As shown previously, V79 cells were devoid of activity towards (+)-anti-BPDE. All the cell lines studied demonstrated appreciable GST activity towards 1-chloro-2,4-dinitrobenzene, but no activity with (-)-anti-BPDE. The latter result suggests that GST Pi is the sole or predominant GST in these cell lines. This was confirmed by HPLC analysis of purified enzymes obtained by affinity chromatography. However, when the catalytic activities of the pure enzymes were determined, all four different GST Pi enzymes were found to be highly capable of conjugating (+)-anti-BPDE with GSH. This observation indicates the existence of an intracellular factor that selectively inhibits conjugation of (+)-anti-BPDE, but not of 1-chloro-2,4-dinitrobenzene in the V79 and CHO cell lines. This new phenomenon seems to be specific for Chinese hamster, since both these cell lines originate from this species.

Primary structures of three Armenian hamster monoclonal antibodies specific for idiotopes and metatopes of the monoclonal anti-fluorescein antibody 4-4-20

This paper reports the complete V γ, V κ, C γ1 and C κ nucleotide and deduced amino acid sequences of two hamster monoclonal anti-metatype antibodies, 3A5-1 and 4A6. These antibodies have been previously characterized in terms of their binding and molecular stabilization properties with liganded murine monoclonal and single-chain antibody 4-4-20 active sites. Also reported are the complete V κ and C κ nucleotide and deduced amino acid sequence of hamster monoclonal anti-idiotype antibody 1F4, which is specific for the unliganded 4-4-20 active site. Oligonucleotide primers based on the 5′ ends of murine variable genes, along with primers specific for murine IgG C γ1 and κ constant region genes, have been used in cDNA and polymerase chain reactions (PCRs) to amplify IgG cDNA from Armenian hamster/mouse hybridomas. The hamster C γ1 and C κ domain sequences are highly homologous to previously reported murine sequences. The anti-idiotype mAb V κ gene demonstrated strong similarity to the murine V κV gene subgroup while the two anti-metatype mAb V κ genes approximated more closely to the murine V κIII gene subgroup. The two anti-metatype mAbs utilized highly homologous V γ genes, with differing HCDR 3 regions, that appeared similar to the murine V γI(a) subgroup. These sequence determinations represent the first primary structures reported for antibodies with anti-metatype activity and are additions to the relatively sparse hamster immunoglobulin genetic database. Results are discussed in terms of 4-4-20 active site specificity and anti-metatype activity, as well as immunoglobulin structural diversity in an anti-Ig immune response.

Novel Monoclonal Antibody Reactive with Thrombin-Sensitive 74-kDa Glycoproteins Present on Platelets and Megakaryocytes Both from Mouse and Rat

A monoclonal antibody (designated 1C2) that reacts only with mouse platelets and megakaryocytes among hematopoietic cells was established by immunizing mouse platelets to an Armenian hamster. 1C2 reactive mouse molecule (1C2 antigen) was a surface glycoprotein with molecular weight of 74 kDa. Side by side comparison revealed that 4A5, a rat monoclonal antibody against mouse platelet, immunoprecipitated the identical molecule to 1C2 antigen. Of particular interest, 1C2 also labeled rat tissues with an identical pattern to that of mouse tissues and recognized a 74-kDa protein from rat platelets. Reactivity of 1C2 to mouse and rat platelets decreased when they were treated with thrombin. Following thrombin treatment of mouse platelets, 1C2 reactive 69-kDa protein appeared in the supernatants. Mouse and rat 1C2 antigens purified on 1C2-coated beads were cleaved by thrombin to generate 69-kDa fragments, establishing that 1C2 antigen is a direct substrate for thrombin. 1C2 is the first antibody to platelets and megakaryocytes of mouse and rat whose reactive molecule is well characterized, i.e., substrate for thrombin. 1C2 can be a useful tool in studying megakaryocytopoiesis and thrombopoiesis in rodent systems.

Characterization of a 50 kDa surface membrane protein on thymic stromal cells as an important factor for early T cell development.

We previously reported that the nude mouse-derived splenic T cell clone, N-9F, exhibits a proliferative response to the SL10.3 thymic epithelial cell clone. In the present study we generated an Armenian hamster mAb, HS9, specific for SL10.3, which inhibited the N-9F's proliferative response to SL10.3. We performed thymocyte repopulation experiments using fetal liver cells and 2'-deoxyguanosine-treated thymic rudiments. After 14 days of culture, donor fetal liver cells proliferated and differentiated to CD4+CD8+ and CD4-CD8+ with some CD4+CD8- cells in the host thymic rudiments. However, most of the thymocytes remained at a CD4-CD8- immature stage in the presence of HS9 and the cell recovery was reduced to 30% of the control. Immunohistostaining and flow cytometry studies revealed that HS9 reacted with stromal cells of fetal thymus at the earliest from day 14 gestation. Neither thymocytes nor lymph node T cells were stained with HS9. HS9 antigen was distributed not only on thymic subcapsular and cortical stromal cells, but also on peripheral B cells in adult mice. The antigen that HS9 detected was found to be a 50 kDa surface membrane protein on thymic stromal cells. On the other hand, the 50 kDa molecule is associated with two other molecules of 80 and 100 kDa on the B cells. These data indicate that the HS9 antigen may have an important role for early T cell development, especially at a stage from CD4-CD8- to CD4+CD8+, and may have some unknown function on B cells.

Novel mAbs reveal potent co-stimulatory activity of murine CD27.

Members of the tumor necrosis factor receptor (TNFR) family are emerging as important molecules implicated in the regulation of proliferation, differentiation and survival of T and B lymphocytes. Among these receptors is CD27, the function of which has thus far only been studied in the human system, where it amplifies the T cell proliferative response induced by TCR triggering. We report here the generation of mAbs to murine CD27, by an efficient method involving the use of transfected Armenian hamster fibroblasts. Previous analysis had already indicated that murine CD27 mRNA is uniquely expressed in lymphoid cells. As determined with one of the newly developed antibodies, murine CD27 is expressed on the great majority of both alpha beta and gamma delta T lymphocytes, on a small population of peripheral B cells, and on a very small subset of B220+ cells in the bone marrow. This distribution largely corresponds to that in the human system. However, unlike human CD27, which is primarily expressed in mature, medullary thymocytes, murine CD27 is found on all thymocytes, except a subset of CD4-CD8- precursors. Upon cross-linking, anti-CD27 mAb amplified the proliferative response of purified T lymphocytes to suboptimal stimulation with concanavalin A at least 4-fold. This indicates that such mAbs can mimick ligand binding and demonstrates that CD27 also acts as a potent co-stimulatory molecule in the murine system.

Clastogenic action of thiotepa on bone marrow cells of the Armenian hamster

Thiotepa (TT) induced chromosomal aberrations in the bone marrow cells of the Armenian hamster, Cricetulus migratorius, in a dose-dependent manner. The maximal clastogenic action was observed 24 h after treatment with thiotepa. Male and female Armenian hamsters were similarly sensitive to the clastogenic action of thiotepa. The lowest effective concentration of thiotepa for Armenian hamsters was 2.5 mg/kg b.w. (6.1% of the LD 50). The results obtained and analysis of the literature data have shown that Armenian hamsters are much more resistant to the clastogenic action of thiotepa than various other species of rodent. This resistance is not due to the metabolic activation rates of chemical mutagen in the liver of Armenian hamsters, since thiotepa is a direct-acting clastogen.