Normal Parental Strain Research Articles

Dual mechanisms diminishing tonic GABAA inhibition of dentate gyrus granule cells in Noda epileptic rats

The Noda epileptic rat (NER), a Wistar colony mutant, spontaneously has tonic-clonic convulsions with paroxysmal discharges. In the present study, we measured phasic and tonic γ-aminobutyric acid A (GABAA) current (I tonic) in NER hippocampal dentate gyrus granule cells and compared the results with those of normal parent strain Wistar rats (WIS). I tonic, revealed by a bicuculline-induced outward shift in holding current, was significantly smaller in NER than in WIS (P < 0.01). The frequency of inhibitory postsynaptic currents (IPSCs) was also significantly lower in NER than in WIS (P < 0.05), without significant differences in the IPSC amplitude or decay time between WIS and NER. I tonic attenuation in NER was further confirmed in the presence of GABA transporter blockers, NO-711 and nipecotic acid, with no difference in neuronal GABA transporter expression between WIS and NER. I tonic responses to extrasynaptic GABAA receptor agonists (THIP and DS-2) were significantly reduced in NER compared with WIS (P < 0.05). Allopregnanolone caused less I tonic increase in NER than in WIS, while it prolonged the IPSC decay time to a similar rate in the two groups. Expression of the GABAA receptor δ-subunit was decreased in the dentate gyrus of NER relative to that of WIS. Taken together, our results showed that a combination of attenuated presynaptic GABA release and extrasynaptic GABAA receptor expression reduced I tonic amplitude and its sensitivity to neurosteroids, which likely diminishes the gating function of dentate gyrus granule cells and renders NER more susceptible to seizure propagation.

Reduction of asialo pathway gangliosides in Escherichia coli-activated peritoneal macrophages from endotoxin hyporesponsive mice

Sialidase-sensitive gangliosides are reduced in endotoxin-activated macrophages from C3H/HeJ mice. Ganglioside variation in mice has been linked to the H-2 complex. The H-2 gene locus is the major histocompatibility complex of the mouse which encodes for major cell surface antigens as well as immune response genes (the Ir genes). To determine whether the ganglioside alteration is a direct pleotropic influence of the Lps defect or whether there is an indirect influence due to the H-2 haplotype, we examined some members of the BXH/Ty set of recombinant inbred mice. The BXH/Ty set was derived by a cross of the normal C75BU6J and the C3H/HeJ strain. The BxH-4/Ty strain was chosen as a hyporesponsive strain and the BxH-14/Ty strain for the normal counterpart. The normal parental strain, C57BI/6J, and the normal BxH-14/Ty strain show patterns consistent with the normal C3H/HeN strain. The reduction of sialidase-sensitive gangliosides (primarily GM1b (cisGM1)) occurs in the endotoxin-hyporesponsive strains, C3H/HeJ and BxH-4/Ty. The hyporesponsive C3H/HeJ parent strain and the normal BXH-14/Ty recombinant strain are H-2 k haplotype whereas the normal C57BI/6J parent strain and the hyporesponsive BXH-4/Ty recombinant strain are H-2 b haplotype. The ganglioside shifts are consistent with a major pleotropic effect of the Lps defect, not H-2 haplotype.

Intrathymic and Extrathymic Tolerance in Bone Marrow Chimeras

Parent-->F1 bone marrow (BM) chimeras provide a useful model for studying self tolerance induction. When prepared with supralethal irradiation (1300 cGy) and conditioned with anti-T cell antibodies, parent-->F1 BM chimeras are devoid of host BM-derived cells; host H-2 expression is apparent in both the intrathymic and extrathymic environments but is limited to non BM-derived cells. When parent-->F1 chimeras are injected with T cells from normal parental strain mice, the expression of host H-2 antigens on nonprofessional APC might be expected to induce tolerance through induction of clonal anergy. In practice, this does not occur. Instead, a small proportion of the injected T cells is induced to proliferate and differentiate into effector cells. Tolerance is not seen. Similarly, tolerance is not apparent when thymectomized parent-->F1 chimeras are given parental strain thymus grafts. These findings suggest that the expression of host H-2 antigens in the post-thymic environment of chimeras is not intrinsically tolerogenic for mature T cells or recent thymic emigrants. Interestingly, post-thymic tolerance does occur when parental strain T cells differentiate in the endogenous thymus of chimeras. Thus, when mature CD8+ cells are prepared from thymus vs lymph nodes (LN) of parent-->F1 chimeras, tolerance to host class I antigens is more marked in LN than thymus; this applies to cytotoxic T lymphocyte (CTL) precursors, generated by limiting dilution analysis. It would appear therefore that many of the host-reactive CTL precursors generated in the thymus of chimeras undergo tolerance induction (deletion or irreversible inactivation) in the post-thymic environment. We suggest that such tolerance is a reflection of a covert form of tolerance induced in the thymus: intrathymic contact with host antigens on thymic epithelial cells (TEC) in chimeras does not delete typical CTL precursors, but these cells are rendered "semi-tolerant". When cultured in vitro in the presence of lymphokines, the cells are able to recover and differentiate into CTL. In vivo, however, the cells recognize antigen in the periphery in the relative absence of lymphokines and the cells die. Although host class I expression on TEC in chimeras deletes only a small proportion of CTL precursors, contact with TEC induces strong tolerance of CD8+ cells in terms of helper-independent proliferative responses in vitro and induction of lethal graft-versus-host disease in vivo. We postulate that these latter responses are controlled by high-affinity T cells, whereas typical CTL generated in LDA are predominantly low-affinity cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Tolerance of CD8+ T cells developing in parent-->F1 chimeras prepared with supralethal irradiation: step-wise induction of tolerance in the intrathymic and extrathymic environments.

Tolerance of CD8+ cells was examined in parent-->F1 bone marrow chimeras (BMC) prepared with supralethal irradiation; host class I expression in the chimeras was limited to non-BM-derived cells. In terms of helper-independent proliferative responses in vitro and induction of graft-vs.-host disease on adoptive transfer, CD8+ cells from long-term chimeras showed profound tolerance to host antigens irrespective of whether the cells were prepared from the thymus or from spleen or lymph nodes. By limiting dilution analysis, cytotoxic T lymphocyte (CTL) precursors specific for host antigens were rare in the extrathymic lymphoid tissues. In the thymus, by contrast, host-specific CTL precursors were only slightly less frequent than in normal parental strain mice. These host-specific CD8+ cells survived when BMC thymocytes were transferred intravenously to a neutral environment, i.e., to donor strain mice. When transferred to further BMC hosts, however, most of the host-reactive cells disappeared. Collectively, the data suggest that tolerance of CD8+ cells in BMC hosts occurs in both the intrathymic and extrathymic environments. In the thymus, contact with host antigens on thymic epithelial cells deletes CD8+ cells controlling helper-independent proliferative responses and in vivo effector functions but spares typical helper-dependent CTL precursors. After export from the thymus, most of the CTL precursors are eliminated after contacting host antigens on stromal cells in the extrathymic environment.

The clonal organization of the squamous epithelium of the tongue.

Knowledge of the kinetics and stem cell localization of the mouse lingual epithelium is largely based on studies using DNA labelling techniques. We have adopted a different approach, using histochemistry for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD). We have deduced clone size and morphology from studies of patch size and distribution in mice heterozygous for G6PD deficiency and from the identification of clonal enzyme loss induced in normal mice by application of a mutagen. Lingual epithelium of female mice (CBA X GPDX) heterozygous for G6PD deficiency showed multiple clearly defined patches of strong or weak enzyme activity, corresponding in intensity to the strong staining uniformly present in the normal parental strain (CBA) or to the weak staining uniformly present in the G6PD deficient parental strain (GPDX). This pattern results from the random suppression of either the paternal or the maternal X chromosome in each cell early in embryonic development, and the subsequent inheritance of X inactivation in daughter cells, giving rise to phenotypic patches each composed of one or more clones. The patch borders intersected the base of the lingual epithelium at small indentations or at the apices of connective tissue papillae; the surface intersection in some cases bisected filiform papillae. Patch width measured in tissue sections at the mid rete ridge level, showed a clear mode close to 40 microns, corresponding very closely to the mode for rete ridge width (i.e. distance between connective tissue papillae). Further evidence for clonal organization was obtained by inducing mutations in the lingual epithelium of CBA mice by topical mutagen application. A few clearly defined patches of enzyme loss were found with a mean diameter of 36 microns. Their morphology was very similar to that of patches in the heterozygous animals. We interpret these patches as clones derived from stem cells with induced somatic G6PD mutations. We conclude that the mouse lingual epithelium is a stem cell epithelium composed of clonal units of about 40 microns diameter, based on the rete ridge structure and that both connective tissue papillae and filiform papillae occur at the junction of two or more epithelial clones.

Generation of T cells with lytic specificity for atypical antigens. I. A mitochondrial antigen in the rat.

F1 rats primed with normal parental strain lymphocyte populations and restimulated in culture with parental lymphoblasts generate potent cytotoxic T cell responses to unusual antigen systems. Here we describe in the Lewis (L)/DA anti-DA combination an antigen system most likely of mitochondrial origin with the following properties: it is transmitted maternally from DA strain females, inherited in an extra-chromosomal manner, restricted by class I RT1Aa major histocompatibility complex gene products, extinguished on target cells treated with chloramphenicol, and its pattern of expression in different rat strains correlates with restriction fragment-length polymorphisms of mitochondrial DNA. Sequence analysis of the rat ND1 gene indicates that the maternally transferred factor in the rat is not a homologue of the maternally transmitted factor responsible for the mitochondrial antigen in mice. In keeping with its inheritance from DA females, this antigen is present on target cells from (DA female x L male)F1 donors and all other F1 combinations derived from DA female parents, but absent from target cells from some F1 combinations (L/DA and Wistar-Furth [WF]/DA) derived from DA strain males. The presence of this antigen in other F1 combinations (Brown Norway [BN]/DA, August 2880 [AUG]/DA, and PVG/DA) indicates that this mitochondrial antigen system is shared by the DA, BN, and PVG strains, but not by the L and WF strains.

Strong T cell tolerance in parent----F1 bone marrow chimeras prepared with supralethal irradiation. Evidence for clonal deletion and anergy.

T cell tolerance induction was examined in long-term H-2-heterozygous parent----F1 chimeras prepared with supralethal irradiation (1,300 rad). Although these chimeras appeared to be devoid of host-type APC, the donor T cells developing in the chimeras showed marked tolerance to host-type H-2 determinants. Tolerance to the host appeared to be virtually complete in four assay systems: (a) primary mixed lymphocyte reactions (MLR) of purified lymph node (LN) CD8+ cells (+/- IL-2); (b) primary MLR of CD4+ (CD8-) thymocytes; (c) skin graft rejection; and (d) induction of lethal graft-vs.-host disease by CD4+ cells. Similar tolerance was observed in chimeras given double irradiation. The only assay in which the chimera T cells failed to show near-total tolerance to the host was the primary MLR of post-thymic CD4+ cells. In this assay, LN CD4+ cells regularly gave a significant antihost MLR. The magnitude of this response was two- to fourfold less than the response of normal parental strain CD4+ cells and, in I-E(-)----I-E+ chimeras, was paralleled by approximately 70% deletion of V beta 11+ cells. Since marked tolerance was evident at the level of mature thymocytes, tolerance induction in the chimeras presumably occurred in the thymus itself. The failure to detect host APC in the thymus implies that tolerance reflected contact with thymic epithelial cells (and/or other non-BM-derived cells in the thymus). To account for the residual host reactivity of LN CD4+ cells and the incomplete deletion of V beta 11+ cells, it is suggested that T cell contact with thymic epithelial cells induced clonal deletion of most of the host-reactive T cells but spared a proportion of these cells (possibly low affinity cells). Since these latter cells appeared to be functionally inert in the thymus (in contrast to LN), we suggest that the thymic epithelial cells induced a temporary form of anergy in the remaining host-reactive thymocytes. This anergic state disappeared when the T cells left the thymus and reached LN.

Fibrinogen concentration in the aqueous humor of buphthalmic rabbits.

Fibrinogen concentration in the aqueous humor of buphthalmic rabbits, AXBU/J, and those of the normal parent strain AX/J are 0.36 +/- 0.14 and 0.08 +/- 0.01 mg/ml, respectively. There was no detectable fibrinogen in the aqueous humor of normal inbred strain III/J rabbits. Among the buphthalmic rabbits, elevated fibrinogen concentration in the aqueous humor was observed in two month old prodromal rabbits, indicating a small leakage of fibrinogen into the anterior chamber before the development of buphthalmia. Protein concentration in the aqueous humor was close to normal, in spite of an elevated level of fibrinogen in the young buphthalmic rabbits. Severe protein leakage was seen in only some of the old buphthalmic rabbits.

Natural Variants of the Cellular Slime Mold Acrasis rosea

SYNOPSIS. Twenty different isolates of the cellular slime mold Acrasis rosea, obtained from diverse sources and geographic regions, were studied to determine similarities and differences in their development and structure in culture and their sensitivity or resistance to selected chemicals incorporated into the culture media. Six different classes of fruiting were defined based on the size, distribution, and type of sorocarps formed on the yeast, Rhodotorula, streaked on agar. In the course of these studies a significant mutant, NC‐18V (variant), developed spontaneously from the wild type, normal parent strain NC‐18N. The mutant differed considerably from all other Acrasis isolates, appeared several times in purified parental cultures, and represents the first laboratory derived variant of A. rosea to be described. Purified strains of the variant (V) and normal (N) cultures were obtained by single‐spore isolation. Normal and variant amebae were mixed in ratios of 10:1 and 100:1 (N:V) and spore and stalk cells were selected from different sorocarps in various regions of the culture plate for analysis. The results of these selection experiments clearly indicate that the individual variant amebae have increased migratory ability and that they develop smaller, morphologically different, and more numerous sorocarps that form at distances further from the food source than NC‐18N. Some isolates of Acrasis no longer were able to fruit and were classified as “non‐fruiters” and a few other isolates formed only a few, small sorocarps on rare occasions. These isolates were mixed together in various combinations of 2 and 3 to screen for cell interaction, but no synergism contributing to fruiting was found. Although fruiting of many A. rosea isolates was inhibited by exposure to continuous light or constant darkness, some “escape”fruiting was noted in certain isolates even when small inocula were used. Single spore isolates of these escape fruiters still fruited in continuous light or dark, but fruiting was always greatly enhanced by a routine 12 hr light : 12 hr dark incubation cycle. It was shown by biochemical studies that actidione, crystal violet, malachite green, ethyl violet, and 5‐fluorodeoxyuridine selectively killed some isolates and permitted a classification of isolates as either sensitive or resistant. In a further study of cell interaction between 2 different sets of Acrasis isolates with contrasting biochemical and morphologic markers the formation of neotypes or recombinants could not be demonstrated.The results of this study clearly indicate the existence of significant variation in A. rosea and the potential for application of these differences to developmental studies.

Modification of the responsiveness of F1 hybrid rats to lymphocytes from parental strain rats.

SummaryAn experimental protocol to modify the susceptibility of F1 hybrid rats to graft‐versus‐host disease produced by parental strain lymphocytes has been described. This procedure entails preliminary exposure of the F1 hybrids to parental strain lymphocytes which have been treated with mitomycin‐C The Susceptibility of F1 hybrids treated in this manner to parental strain lymphocyte varies with the interval which has elapsed before the injection of normal parental strain lymphocytes. It appears that, in order to afford protection, the cells of the preliminary inoculum are required to engage the lymphoid cells of the host in an allogeneic interaction and that F1 hybrid unresponsiveness on re‐exposure to parental strain cells reflects unresponsiveness of host lymphocytes. Concomitant modification of the susceptibility of F1 hybrids to lymphocytes from donors of the Other parental strain was observed alter preliminary exposure to cells of one parental strain.

Activation of T and B thymus cells to recognize histocompatibility antigens

Lethally irradiated (A × CBA) F 1 or (A × C57BL/6) F 1 mice were injected with 10 7 A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F 1 hybrid spleen cells) into corresponding unirradiated F 1 hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F 1 mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F 1 hosts not containing the antigens against which activation had been directed.

Proliferation of activated thymocytes in mixed lymphocyte reactions.

When parental strain thymocytes were transferred to heavily irradiated F1 mice large numbers of blast lymphocytes could be recovered from the thoracic duct 4 days later. This population of thymus derived thoracic duct lymphocytes (T-TDL) comprised 95–100% of donor cells (1, 2). When T-TDL were transferred to further irradiated F1 hybrid mice, syngeneic with the primary hosts, the cells entered a brief phase of DNA synthesis, reaching a peak on days 2 and 3, and falling to very low levels by day 4. In contrast, DNA synthesis was very low when T-TDL were transferred to “third party” F1 mice and was not detected in mice syngeneic with the original cell donors. This pattern differed from that observed with normal parental strain thoracic duct lymphocytes (TDL) in that TDL responded equally in the two hybrid strains and did not reach high levels of DNA synthesis until day 4 (3). These observations suggested that T-TDL, unlike TDL, could only proliferate in response to those antigens which initially provoked their formation. This conclusion was supported by the high degree of specificity manifested by T-TDL in their capacity to reject tumour and skin allografts and to lyse appropriate target cells in vitro (3).

The effect of presensitization of parental donors on graft-versus-host disease in irradiated F 1 hybrid mice.

SummaryThe GVH disease in 1C3F1 hybrid mice following 500 R X-irradiation and injection of normal parent strain spleen cells is mild in terms of mortality, signs, and tissue pathology. Presensitization of the donor with spleen cells of the reciprocal parent strain, however, results in a highly lethal GVH disease. The timing of presensitization before donor use is an important factor in the severity of the resulting GVH disease. Presensitization of the donor seems to require more than 3 days, is near maximum effectiveness at 7 days, persists at this level for many months, and gradually falls off through the life of the donor. After 24 months, sensitized donors still are more effective in producing GVH disease than controls of comparable age.

Histocompatibility antigens in the rat: the AS2 strain in relation to the AS, BS and HS strains.

SummaryThe origin of the inbred albino rat strain AS2 is described. With the exception of weak male → female incompatibility, the strain appears to be isohistogenic and homozygous at all loci determining histocompatibility antigens. The weak antigen distinguishing males from otherwise isohistogenic females is identical in the AS2 and HS strains. The AS2 strain possesses at least one histocompatibility antigen in common with each of strains AS, BS and HS. The AS. BS and HS strains possess identical alleles at a major histocompatibility locus at which they differ from the AS2 strain. Donor‐host disparity at this locus regularly leads to the development of local graft versus host reactions following the injection of 50 × 106 normal parental strain spleen cells beneath the renal capsule of F1 hybrids. Where donor and host are compatible at this locus, despite disparity at up to 3 other loci determining strong antigens, local graft versus host reactions do not occur following normal spleen cell transfer, although they occur somewhat irregularly if specifically sensitised spleen donors are used. It therefore seems likely that this major locus is the Ag‐B locus. Mean skin graft survival times are only slightly shorter in Ag‐B incompatible combinations than in those combinations which are compatible at Ag‐B hut differ in respect of other strong histocompatibility antigens. Parental strain → F2 hybrid skin grafts show the closely related AS and AS2 strains to differ from each other at an estimated 4‐5 histocompatibility loci, one of which is the Ag‐B locus.

The development of resistance to glycarbylamide and 2-chloro-4-nitrobenzamide in Eimeria tenella in chicks.

A twofold increase in resistance to glycarbylamide was induced in a strain of Eimeria tenella in chicks. This strain remained susceptible to amprolium, nicarbazin, nitrofurazone, zoalene, 3,5-dinitrobenzamide, 2-chloro-4-nitrobenzamide (M &amp; B 5921) and spiramycin.At least an eightfold resistance to 2-chloro-4-nitrobenzamide (M &amp; B 5921) was developed in another strain of E. tenella. This strain was also resistant to nitrofurazone, zoalene and 3,5-dinitrobenzamide, but not to glycarbylamide, amprolium, nicarbazin and spiramycin.A single test showed no transfer of drug-resistance when the two resistant strains were given simultaneously to the same birds.When a small number of parasites of a glycarbylamide-resistant strain of E. tenella was introduced into a larger inoculum of the normal parent strain, the resistant individuals appeared to diminish in number during passages through untreated chicks.I wish to thank Mrs B. M. Mitchell, Miss C. A. Hitchcock and Miss J. Watkins for technical assistance at various stages of the work.

Observations on the dehydrogenase activity of normal and drug-resistant strains of Trypanosoma rhodesiense

1. 1. A method is described for the photometric estimation of the dehydrogenase activity at 25 °C of washed suspensions of Trypanosoma rhodesiense, and the conditions necessary for reproducible results are outlined. 2. 2. Rabbit serum had a marked effect on the glucose dehydrogenase activity of washed T. rhodesiense suspensions; activity was promoted by concentrations as low as 0.0004% in the presence of M/100 glucose. 3. 3. A strain of T. rhodesiense fully resistant in mice to ‘Melarsen’ (sodium p-melaminylphenylarsonate) differed markedly from the normal parent strain in respect of activation of six out of ten substrates tested.