T-lymphocyte heterogeneity in the rat: Separation of distinct rat T-lymphocyte populations which respond in syngeneic and allogeneic mixed lymphocyte reactions

These experiments were designed to determine if separate subpopulations of T cells were involved in the syngeneic and allogeneic mixed lymphocyte reaction. Rat lymph node T cells were separated into W3/25⁺ and W3/25^? subpopulations by panning with the monoclonal antibody W3/25 and tested for their ability to proliferate in both syngeneic (SMLR) and allogeneic (MLR) mixed lymphocyte responses, as well as to develop cytotoxicity against allogeneic, syngeneic, and trinitrophenol (TNP)-modified syngeneic targets. The W3/25⁺ T cells reacted strongly in the SMLR and the MLR whereas the W3/25^? fraction proliferated only in response to allogeneic stimulation and with a kinetic pattern distinct from W3/25⁺. Furthermore, addition of W3/25 monoclonal antibody directly to the cultures was shown only to inhibit the proliferation of the W3/25⁺ T-cell fraction. The W3/25^? subpopulation contained cytotoxic T cells (CTLs) against both allogeneic determinants and TNP-modified self. However the requirements for the activation of allospecific CTLs were distinct from those for CTLs for TNP-self in that W3/25^? allospecific CTLs required no detectable help from W3/25⁺ T cells but generation of the CTL response against TNP-self required the presence of W3/25⁺ helper T cells (Th). These data suggest that in the rat, there exist subsets of T cells recognized by their cell surface phenotype that distinguish between self and nonself determinants and the requirements for activation are different for each of these populations.     

Cloning and activation of the Syrian hamster neu proto-oncogene

Point mutations of the Syrian hamster neu proto-oncogene have been observed in the transmembrane domain of N-nitroso-N-ethylurea(ENU)-induced neurofibromas, Genomic DNA derived from tumor tissue showed transforming activity in an NIH 3T3 assay and a cDNA clone of the hamster neu gene, containing the entire protein-coding region, was isolated from a transformant cDNA library. The encoded product is 92 and 88% homologous to the rat neu and the human c-erbB-2, respectively. The product of the mutated hamster neu gene showed increased autophosphorylation of Tyr residues.     

Separate domains of KAR1 mediate distinct functions in mitosis and nuclear fusion

The yeast KAR1 gene is essential for mitotic growth and important for nuclear fusion. Mutations in KAR1 prevent duplication of the spindle pole body (SPB), and affect functions associated with both the nuclear and cytoplasmic microtubules. The localization of hybrid Kar1-lacZ proteins, described elsewhere (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press), suggest that the protein is associated with the SPB. In this paper, we report a deletion analysis demonstrating that the mitotic and karyogamy functions of KAR1 are separate and independent, residing in discrete functional domains. One region, here shown to be essential for mitosis, coincided with a part of the protein that is both necessary and sufficient to target Karl-lacZ hybrid proteins to the SPB (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Complementation testing demonstrated that deletions in this interval did not affect nuclear fusion. A second region, required only for karyogamy, was necessary for the localization of a Kar3-lacZ hybrid protein to the SPB. These data suggest a model for the roles of Kar1p and Kar3p, a kinesin-like protein, in nuclear fusion. Finally, a third region of KAR1 was found to be important for both mitosis and karyogamy. This domain included the hydrophobic carboxy terminus and is sufficient to target a lacZ-Kar1 hybrid protein to the nuclear envelope (Vallen E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Altogether, the essential mitotic regions of KAR1 comprised 20% of the coding sequence. We propose a model for Kar1p in which the protein is composed of several protein-binding domains tethered to the nuclear envelope via its hydrophobic tail.     

Two phenotypically distinct subsets of spleen dendritic cells in rats exhibit different cytokine production and T cell stimulatory activity

We recently reported that splenic dendritic cells (DC) in rats can be separated into CD4(+) and CD4(-) subsets and that the CD4(-) subset exhibited a natural cytotoxic activity in vitro against tumor cells. Moreover, a recent report suggests that CD4(-) DC could have tolerogenic properties in vivo. In this study, we have analyzed the phenotype and in vitro T cell stimulatory activity of freshly isolated splenic DC subsets. Unlike the CD4(-) subset, CD4(+) splenic DC expressed CD5, CD90, and signal regulatory protein alpha molecules. Both fresh CD4(-) and CD4(+) DC displayed an immature phenotype, although CD4(+) cells constitutively expressed moderate levels of CD80. The half-life of the CD4(-), but not CD4(+) DC in vitro was extremely short but cells could be rescued from death by CD40 ligand, IL-3, or GM-CSF. The CD4(-) DC produced large amounts of the proinflammatory cytokines IL-12 and TNF-alpha and induced Th1 responses in allogeneic CD4(+) T cells, whereas the CD4(+) DC produced low amounts of IL-12 and no TNF-alpha, but induced Th1 and Th2 responses. As compared with the CD4(+) DC that strongly stimulated the proliferation of purified CD8(+) T cells, the CD4(-) DC exhibited a poor CD8(+) T cell stimulatory capacity that was substantially increased by CD40 stimulation. Therefore, as previously shown in mice and humans, we have identified the existence of a high IL-12-producing DC subset in the rat that induces Th1 responses. The fact that both the CD4(+) and CD4(-) DC subsets produced low amounts of IFN-alpha upon viral infection suggests that they are not related to plasmacytoid DC.     

Novel immunoregulatory functions of phenotypically distinct subpopulations of CD4+ cells in the human neonate.

Although normal numbers of CD4+ T cells are present in the human neonate, cord blood CD4+ cells are deficient in their ability to provide help for antibody production. In the present studies, we have examined the cellular basis for this functional deficit by analyzing the phenotypic properties and immunoregulatory functions of the subsets of cord blood CD4+ cells defined by anti-CD45RA mAb. In contrast to CD4+ cells from adults, greater than 90% of cord blood CD4+ cells expressed the CD45RA, CD38, and Leu-8 membrane Ag. When neonatal CD4+ cells were cultured with adult B cells and PWM or anti-CD4+ mAb, no helper function was apparent. However, when the small number of CD4+CD45RA- cells in cord blood were purified and similarly analyzed, helper activity comparable to that of adult CD4+CD45RA- cells was found. This helper function was profoundly suppressed by the presence of even small numbers of cord blood (but not adult) CD4+CD45RA+ cells. Irradiation of mitomycin C treatment of neonatal CD4+CD45RA+ cells abrogated their suppressor activity, but did not induce helper capability. However, after activation with PHA and culture in IL-2, cord blood CD4+CD45RA+ cells lost their suppressor activity and acquired the ability to provide help for B cell differentiation. This functional maturation was accompanied by their conversion to the CD4+CD45RA- phenotype. Thus, whereas cord blood CD4+CD45RA+ and CD4+CD45RA- cells share certain properties with the analogous subsets in adults, our data show that the dominant immunoregulatory function of cord blood CD4+ cells is suppression mediated by CD4+CD45RA+ (and CD38+) cells. In view of these phenotypic and functional differences between neonatal and adult CD4+CD45RA+ cells, we propose that "naive" CD4+CD45RA+ cells undergo age-related maturational changes that are unrelated to their postulated activation-dependent post-thymic differentiation into CD4+CD45RA- "memory" cells capable of helper functions.     

IL-6/BSF-2 functions as a killer helper factor in the in vitro induction of cytotoxic T cells

rIL-6/B cell stimulatory factor 2 was found to augment CTL generation from mature as well as immature human T cells stimulated with UV-treated allogeneic cells. rIL-6 also acted on peanut agglutinin-positive murine thymocytes and Lyt-2-positive splenic T cells to give rise to CTL. rIL-6 alone could not induce CTL generation, the presence of IL-2 during the early phase of culture period was found to be essential for the IL-6 activity in the induction of CTL. The effect of rIL-6 was not mediated by the induction of IL-2 inasmuch as rIL-6 did not augment IL-2 production in MLC and anti-IL-2 antibody could not neutralize IL-6 activity. rIL-6 augmented CTL generation even when added 72 h after the initiation of cultures. The enhancing activity of rIL-6 could be neutralized with anti-IL-6 antibodies even when added 72 h after the initiation of cultures. The present data indicate that IL-6 acts in the late phase of CTL generation.     

MHC-unrestricted cytotoxic and proliferative responses of two distinct human gamma/delta T cell subsets to Daudi cells.

Human V gamma 9/V delta 2 T cells, the major subset of gamma/delta T cells in peripheral blood of adults, mediate proliferative and cytotoxic responses to Daudi Burkitt's lymphoma cells without previous in vitro exposure to Daudi. Our experiments show that some gamma/delta T cells coexpressing V gamma 9 and V delta 1 genes also react to Daudi cells in cytotoxic and proliferative assays. Expression of V gamma 9 is not sufficient for the recognition of Daudi cells because most gamma/delta T cells expressing V delta 1 paired with V gamma 9 or other V gamma genes neither kill Daudi cells nor proliferate to Daudi. V gamma 9/V delta 2 T cells do not proliferate to other cell lines such as K562 or Molt4 that are sensitive to MHC-unrestricted cytolysis by NK cells and by most IL-2-activated gamma/delta T cell clones. Cold target inhibition assays demonstrate that Daudi cells are stronger inhibitors than K562 and Molt4 of MHC-unrestricted lysis by V gamma 9/V delta 2 clones. However, cold Daudi cells are relatively weaker inhibitors of MHC-unrestricted lysis by NK cell clones, most gamma/delta T cell clones expressing V delta 1 and alpha/beta T cell clones. Thus, recognition by V gamma 9/V delta 2 T cells and certain V gamma 9/V delta 1 T cells of Daudi appears to involve a specific triggering pathway that is distinct from recognition by these gamma/delta T cells of Molt4, K562, and other target cells. NK cell clones and most other gamma/delta and alpha/beta T cell clones derived from the same normal volunteer blood donors do not show this specific interaction with Daudi cells. These data show that distinct subsets of human gamma/delta T cells recognize Daudi cells and support the idea that the gamma/delta TCR may be directly involved.     

Urethane and N-nitrosodiethylamine are mutagenic for the Syrian hamster fetus

Urethane and N-nitrosodiethylamine are soluble environmental carcinogens that initiate tumors transplacentally, but have a mixed history of effectiveness in mutagenesis assays in vitro or in vivo with adult rodents. To test for their transplacental mutagenicity, Syrian hamster fetuses at 12 days in gestation were exposed transplacentally to urethane or N-nitrosodiethylamine at 0.5 or 1.0 mM/kg. The fetal cells were isolated on day 13 of gestation and tested for diphtheria toxin resistance as a mutation marker. Both compounds were significantly mutagenic, at both doses, causing 6- to 20-fold increases in mutations compared with controls. Compared with N-nitrosodiethylamine, urethane was somewhat more effective as a mutagen with a more marked dose–response. These results are consistent with mutagenesis as part of the mechanism of transplacental carcinogenicity of urethane and N-nitrosodiethylamine.     

Breeding history and olfactory discrimination in the Syrian hamster

The ability of outbred female Syrian hamsters to differentially respond to urine samples obtained from either inbred or outbred Syrian males was examined. The odors obtained from the males were introduced into a Y-maze olfactometer. On the basis of time spent in proximity to the stimuli, estrous females evidenced a preference for the urine of the outbred males. The same females when diestrus exhibited no apparent preferences. It is possible that the ability of females to make such subtle discriminations may impact on mate selection leading to positive assortment.     

Naloxone antagonism of diazepam-induced feeding in the Syrian hamster

Three experiments investigated the effects of the intragastric administration of the benzodiazepine diazepam on feeding in non-deprived Syrian hamsters ($\text{mesocricetus auratus}$). In the first experiment diazepam (0, 0.5, 1.0, 2.0, and 4.0 mg/kg) produced dose dependant increases in feeding. 4.0 mg/kg of diazepam produced significantly more feeding than the other doses tested and the lowest dose tested (0.5 mg/kg) produced a significant increase in feeding. In the second experiment naloxone (10 mg/kg) partially antagonized the effect of 4 mg/kg of diazepam on feeding. In the third experiment the ability of naloxone (0.1, 1.0, 5.0, 10.0 or 20 mg/kg) to reduce feeding produced by either 4 mg/kg or 2 mg/kg of diazepam was tested. Naloxone partially antagonized the effects of 4 mg/kg of diazepam on feeding in a dose dependant manner. While 2 mg/kg of diazepam produced significantly less feeding than 4 mg/kg, naloxone did not antagonize the effect of 2 mg/kg on feeding. The results suggest that two mechanisms are involved in diazepam-induced feeding in hamsters. The high dose of diazepam may produce increased feeding by activating the endorphin system while the low dose of diazepam produces increased feeding via a naloxone insensitive mechanism.     

Reproductive effects of olfactory bulbectomy in the Syrian hamster

The effects of olfactory bulbectomy on circulating gonadotropin, prolactin and testosterone levels and on the testicular and pituitary responses to shortening of day length were studied in Syrian hamsters. Adult animals maintained on a 14L:10D cycle were sham-operated or sustained bilateral radical olfactory bulbectomies by aspiration to remove the main and accessory olfactory bulbs and the adjacent regions of the anterior olfactory nucleus. They were then maintained either on the long photoperiod or housed on a 10L:14D cycle. Testicular length was measured at weekly intervals over a 5-mo period. Sham-operated controls exhibited the normal pattern of testicular regression and eventual recrudescence on the short photoperiod. Testicular regression was significantly reduced in bulbectomized animals. Many of these animals showed no regression; others exhibited a reduced degree and/or shortened duration of regression. Serum levels of follicle-stimulating hormone (FSH) were substantially elevated in bulbectomized males maintained in long days. Their serum levels of luteinizing hormone (LH), prolactin and testosterone remained within the range for shams on long photoperiod. In short days, the bulbectomized animals showed the normal, pronounced decline in circulating prolactin levels. Serum FSH and LH levels also showed substantial declines, but the FSH levels were not reduced below the range for controls in long days, and the decline in LH levels was not as great as that for controls in short days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glomerular lipidosis in a Syrian hamster of the APA strain

Spontaneous glomerular lipidosis was found in a 12-week-old male Syrian hamster of the APA strain. Lipids in the glomeruli were observed as droplets in a prominently expanded mesangial area and as emboli in a dilated capillary lumen. Lipid deposition was also, but less often, detected in tubular epithelial cells and interstitial cells around the lipid-laden glomeruli. This case of glomerular lipidosis was considered to be closely related to hyperglycaemia and hyperlipidaemia.     

Pathology of experimental Babesia microti infection in the Syrian hamster

Pathologic changes produced after 4 weeks of infection by Babesia microti in Syrian hamsters are described and compared to babesiosis of humans. Following intraperitoneal inoculation, both intravascular and extravascular hemolysis developed. Up to 70% of red blood cells were parasitized. The principal morphologic abnormalities were an increase in extramedullary hematopoiesis and hyperplasia of the mononuclear phagocytic cells of the red pulp manifested grossly as splenomegaly, marked renal tubular hemosiderosis and hypertrophy of Kupffer cells. The disease was not fatal to any hamsters during the 4 week study. The clinical signs and lesions were less severe than fatal babesiosis of asplenic humans and similar to severe, but nonfatal disease in spleen intact humans. The hamster may serve as an animal model for the studying the pathophysiology of human babesiosis and for studying potential chemotherapeutic agents.     

Oxidative metabolites of diethylstilbestrol in the fetal Syrian golden hamster

14C-Diethylstilbestrol was administered orally, intraperitoneally, and intrafetally to 15-day pregnant hamsters at a dose of 20 mg/kg body weight, and the radioactivity was determined in the fetus, placenta, and maternal liver after 6 hours. Significant amounts of radioactivity were found in these tissues in every case, indicating maternal-fetal and fetal-maternal transfer of diethylstilbestrol. Part of the radioactivity found in the tissues could not be extracted even after excessive washing. This implied the presence of reactive metabolites. In the fetal and placental extracts, eight oxidative metabolites of diethylstilbestrol were identified by mass fragmentography as hydroxy- and methoxy-derivatives of diethylstilbestrol, pseudodiethylstilbestrol, and dienestrol. The presence of oxidative metabolites in the hamster fetus and the covalent binding to tissue macromolecules are possibly associated with the fetotoxic effects of diethylstilbestrol.     

Coronary artery anomalies and aortic valve morphology in the Syrian hamster

In the Syrian hamster, anomalies in the origin of the left coronary artery are significantly associated with the bicuspid condition of the aortic valve. In this species, bicuspid aortic valves are expressions of a trait, the variation of which takes the form of a phenotypic continuum, ranging from a tricuspid aortic valve with no commissural fusion to a bicuspid aortic valve with the aortic sinuses located in ventrodorsal orientation and devoid of any raphe. The intermediate stages of the continuum are represented by tricuspid aortic valves with a more or less extensive fusion of the ventral commissure and bicuspid aortic valves with a more or less developed raphe located in the ventral aortic sinus. The present study was designed to decide whether there is a gap between tricuspid and bicuspid aortic valves regarding the incidence of coronary artery anomalies, or whether this incidence varies according to the different tricuspid and bicuspid morphotypes of the continuum. The study was carried out in Syrian hamsters belonging to a single inbred family with a high incidence of tricuspid aortic valves with fusion of the ventral commissure, bicuspid aortic valves, and anomalies in the origin of the left coronary artery, i.e. single right coronary artery ostium in aorta, anomalous origin of the left coronary artery from the pulmonary artery, and anomalous origin of the left coronary artery from the dorsal aortic sinus. The specimens were examined by means of a stereomicroscope and, in several cases, scanning electron microscopy was also used. The relationships between anomalous coronary artery patterns and aortic valve morphologies were tested using a logistic regression model. The results obtained indicate that there is no discontinuity between tricuspid and bicuspid aortic valves regarding the incidence of coronary artery anomalies. The probability of occurrence of anomalous coronary artery patterns increases continuously according to the deviation degree of the aortic valve from its normal (tricuspid) design. The present findings suggest that in the Syrian hamster, the morphogenetic mechanisms involved in the formation of congenital anomalous aortic valves and anomalies in the origin of the left coronary artery, respectively, are strongly related from an aetiological viewpoint.