The immune system preferentially clears Theiler's virus from the gray matter of the central nervous system.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.     

Effect of diabetes mellitus on mouse pre-implantation embryo development.

Fifteen spontaneously diabetic, non-obese mice (NOD strain), 17 non-diabetic NOD mice (in which diabetes had not yet developed) and 9 diabetic NOD mice were treated with insulin. All animals were superovulated with 5 iu of pregnant mares' serum gonadotrophin followed 48 h later by 5 iu human chorionic gonadotrophin (hCG) and mated overnight with NOD males of proven fertility. To assess in-vitro and early in-vivo development, 23 NOD mice were killed 72 h after hCG treatment. Embryos were recovered from oviduct flushings and cultured in Ham's F-10 medium with 0.1% bovine serum albumin at 37 degrees C in an atmosphere of 5% O2, 5% CO2, and 90% N2. Development was assessed at intervals of 24 h for 72 h. Compared with embryos from non-diabetic NOD mice (n = 81), embryos from diabetic NOD mice (n = 68) demonstrated marked impairment in growth assessed by distribution of developmental stages at each observation period (24, 48, 72 h, all P less than 0.001) and by overall rates of progression of developmental stages (P less than 0.01). In diabetic NOD mice treated with insulin, embryo development (n = 7) was not significantly different from that of embryos from non-diabetic NOD mice (n = 81), but was significantly faster than in embryos from diabetic NOD mice not treated with insulin (n = 68) (P less than 0.001, for all periods, overall rate P less than 0.01). To assess late in-vivo growth, 18 NOD mice were killed 120 h after hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferation of PHA-stimulated lymphocytes measured by combined autoradiography and sister chromatid differential staining

Lymphocyte proliferation in culture was studied by combined [³H]TdR incorporation and sister chromatid differential staining. The majority of 1st division metaphases in a 72 h culture commenced DNA synthesis after 48 h and had a cell cycle of less than 24 h. A small proportion of cells from some donors commenced DNA synthesis between 24–30 h and had cell cycle times of up to 48 h. Although many cells entered DNA synthesis at the same time, they showed marked asynchrony in the length of their cell cycle, with some completing one, some two and others three cell cycles in the 72 h culture period. The time taken for cells to enter S following stimulation with PHA ranged from 24 to 48 h and there was considerable variation between donors in the number of fast and slow responding cells.     

Neutralizing anti-IL-10 antibody blocks the protective effect of tapeworm infection in a murine model of chemically induced colitis

There is increasing evidence that parasitic helminth infection has the ability to ameliorate other disease conditions. In this study the ability of the rat tapeworm, Hymenolepis diminuta, to modulate dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice is assessed. Mice receiving DNBS (3 mg intrarectally) developed colitis by 72 h after treatment. Mice infected 8 days before DNBS with five H. diminuta larvae were significantly protected from the colitis, as gauged by reduced clinical disease, histological damage scores, and myeloperoxidase levels. This anticolitic effect was dependent on a viable infection and helminth rejection, because no benefit was observed in mice given killed larvae or in infected STAT6 knockout mice or rats, neither of which eliminate H. diminuta. The anticolitic effect of H. diminuta was associated with increased colonic IL-10 mRNA and stimulated splenocytes from H. diminuta- plus DNBS-treated mice produced more IL-10 than splenocytes from DNBS-only treated mice. Coadministration of an anti-IL-10 Ab blocked the anticolitic effect of prophylactic H. diminuta infection. Also, mice infected 48 h after DNBS treatment showed an enhanced recovery response. Finally, using a model of OVA hypersensitivity, we found no evidence of concomitant H. diminuta infection enhancing enteric responsiveness to subsequent ex vivo OVA challenge. The data show that a viable infection of H. diminuta in a nonpermissive system exerts a profound anticolitic effect (both prophylactically and as a treatment) that is mediated at least in part via IL-10 and does not predispose to enhanced sensitivity to bystander proteins.     

Cytokine production induced by marine algae lectins in BALB/c mice splenocytes

Marine algae can serve as sources of bioactive compounds and currently have been shown their potential biological and pharmaceutical applications. Marine algae lectins have been shown to be effective at controlling inflammatory processes. This work aimed to analyze the immunostimulatory properties of lectins from the marine algae Solieria filiformis (SfL), Pterocladiella capillacea (PcL) and Caulerpa cupressoides (CcL). This analysis was performed on BALB/c mouse splenocytes by measuring cytokine and nitric oxide production and cellular damage using tests of cytotoxicity and cell viability. These lectins were not cytotoxic (1-100 μg/mL), and were not able to induce IFN-γ and IL-2 production. IL- 10 production was induced at high levels by all lectins tested. Treatment with SfL induced IL-6 production at higher levels at all experimental times, whereas treatment with PcL and CcL induced higher levels only in 24 and 72 h. Treatment with SfL did not result in nitrite oxide production, whereas treatment with PcL or CcL was able to induce nitrite release at high levels (after 24, 48 and 72 h). Lesser cellular damage (5%) was observed in splenocytes treated with these lectins (10 μg/mL). Thus, the lectins from these algae were not cytotoxic, promoted increased in cell viability and induced Th2 immune responses in mouse splenocytes, indicating that they have anti-inflammatory effects.     

Factors affecting the yield of viable embryos by superovulated Holstein-Friesian cows

GnRH treatment (250 ug) 48 h after prostaglandin F(2alpha) in 40 superovulated cows induced a release of LH (increment > 5 ng/ml) in only 13 of the older cows. Eleven of these cows did not yield viable embryos. Thirty-two of 75 cows had preovulatory surge levels of LH 48 h after prostaglandin treatment. Plasma progesterone concentrations were determined in 140 cows at the time that superovulation was initiated. Eighty-four of these donors were superovulated with 40 mg of FSH and 56 donors with 48 mg of FSH. There was no relationship (P > 0.05) between the concentration of progesterone at the start of superovulation with either ovulation rate determined by palpation per rectum or the number of viable embryos per flush. These parameters were also unaffected (P > 0.05) by age of the donor or the dose of FSH. In another group of donors, treatment with 40 mg FSH was compared over a 3-d (n = 28) and a 4-d (n = 18) interval. The donors treated with FSH over a 3-d period had similar ovulation rates but yielded less viable embryos (1.5 v 5.8, P < 0.05). The fertility rate of 33 cows, inseminated 60 and 72 h after prostaglandin, was comparable to the fertility rate of 18 cows inseminated at 60, 72 and 84 h after prostaglandin treatment.     

Transfer of diabetes from prediabetic NOD mice to NOD-SCID/SCID mice: association with pancreatic insulin content.

Splenocytes from prediabetic female NOD mice can transfer diabetes to NOD-SCID mice. Whereas the kinetics of disease transfer was shown to be a function of the age of donor splenocytes, information is scarce as to how the stage of autoimmune disease, as evaluated by pancreatic insulin content, is related to the diabetogenic potency of splenic T-cells. We therefore determined individual diabetes transfer times after an i. v. injection of splenocytes from prediabetic NOD mice of different ages into female NOD-SCID mice in relation to the diabetes incidence in NOD donor mice and their pancreatic insulin contents. Three groups (n = 8) of NOD mice aged 5, 11, and 17 weeks (wk) underwent splenectomy and hemipancreatectomy. After that, 10x10 (6) splenocytes either pooled from all donor NOD mice of the different age groups or individually from single donor mice were transferred to groups of four 6-week-old NOD-SCID mice, respectively, in two sets of experiments. Insulin was extracted from the resected hemipancreas, and the insulin content was determined by a RIA. Diabetes in the NOD-SCID cohort occurred after a mean time of 126 days after transfer of pooled splenocytes from 5-week-old NODs, after 68 days (transfer from 11-week-old NODs), and after a mean time of 43 days (transfer from 17-week-old NODs, 5 vs. 11 wk: p < 0.02, 11 vs. 17 wk: p < 0.001). Individual time to diabetes positively correlated with diabetes transfer times in NOD-SCID recipients (p < 0.0001) in the 17-week-old NOD mice, confirming previous diabetes transfer studies in hemi-pancreatectomized NOD mice. Furthermore, individual insulin concentrations in 17-week-old NOD mice also positively correlated to diabetes transfer times in recipient mice (p < 0.0001). No such correlations for these parameters were seen for the 5 and 11-week-old NOD mice (time to diabetes: 11 wk, p = 0.14, 5 wk, p = 0.75; insulin content: 11 wk, p = 0.81, 5 wk, p = 0.14). These data suggest that destructive T-cell activity increases during the course of islet autoimmunity. The immune response seems to be programmed for beta-cell destruction just before diabetes onset. This is the only time that pancreatic insulin content predicts the impending onset of diabetes.     

Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splenocytes and bone marrow cells from T-cell deficient Nu/Nu mice secrete interleukin 3 activity after stimulation in vitro

We show here that the combination of Concanavalin A (Con A), phorbol myristate acetate (PMA), and Ionomycin (Iono) reproducibly stimulated splenocytes from Nu/Nu mice and bone marrow cells from both normal and Nu/Nu mice to secrete interleukin 3 (IL-3) in vitro. IL-3 was measured by its property of supporting the growth of four different clones known to grow only in IL-3. None of the agents indicated above nor several other types of stimuli tested could induce the cells to secrete IL-3 activity. IL-3 activity from induced cells of either tissue was detected after 24 hr of culture, peaked at 48 hr and either declined by 72-96 hr of culture (bone marrow cells) or remained relatively constant through the 4-day culture period (splenocytes). The cells participating in the production of IL-3 activity in Nu/Nu spleen were THY1+, L3T4-, LyT2-, B-220-, J11d-, Ia-, and those in the marrow from either normal or Nu/Nu mice were THY1+, J11d+, L3T4-, LyT2-, B-220-, Ia-. Finally, we present evidence that Ia-positive cells negatively regulate the production of IL-3 activity by both splenocytes and marrow cells. We conclude that Nu/Nu splenocytes and bone marrow cells from both normal and Nu/Nu mice can secrete IL-3 activity after proper stimulation in vitro and that such property is negatively regulated by Ia-positive cells.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice

In order to gain more insight into mechanisms operating on the haematopoietic activity of the T-cell-derived cytokine, interleukin-17 (IL-17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL-17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL-6, IL-10, IGF-I, IFN-gamma and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL-17 did not affect granulocyte-macrophage (CFU-GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU-E) progenitors was increased at 48 h, while the number of mature erythroid (CFU-E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL-17 also increased IL-6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin- progenitor cells, demonstrated a slightly enhancing effect of IL-17 on CFU-GM and no influence on BFU-E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL-17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL-17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU-E found at the same time indicate that IL-17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.     

Pertussis vaccine as an inducer of suppressor cells inhibiting the local graft versus host reaction

The formation of nonspecific suppressor cells inhibiting the development of the graft versus host (GvH) reaction is shown to occur in the spleen of CBA mice after the injection of pertussis vaccine. 2 splenocyte populations have been found to possess suppressing activity: one of them comprises the splenocytes sensitive to anti-theta serum, carrying no receptors to IgG and IgM on their surface and incapable of adherence to the plastic surface; the splenocytes in the other population adhere to the plastic surface. During incubation and in vitro contact with the allogenic cells of intact donors the suppressor splenocytes release soluble mediators capable of suppressing the GvH reaction.     

Branched-chain amino acid-enriched diet: effects on insulin secretion and cellular immune aggression

Several reports have demonstrated that high-protein diets may have beneficial effects on experimental models of diabetes and have raised the possibility that branched-chain amino acids could play a role in these protective effects. We investigated the effect of a normoproteic, branched-chain amino acid-enriched diet (experimental diet) on insulin secretion from C57BL/6N mice transferred with splenocytes from diabetic syngeneic donors. Mice previously fed with the experimental or control diet received three intraperitoneal injections, every other day, of 5 x 107 viable mononuclear splenocytes obtained from control or diabetic donors. Results showed that mice fed with the experimental diet and transferred with "diabetic" splenocytes presented: i) normoglycemia, and (ii) significantly higher levels in both phases of glucose-induced insulin secretion and normal values of arginine-glucose-induced insulin secretion. To evaluate the in vitro cellular immune aggression, dispersed mouse islet cells were co-cultured with splenocytes from syngeneic diabetic mice. First, dispersed islet cells from mice on the experimental or control diet were co-cultured with splenocytes from control or diabetic mice on a commercial diet. In the presence of "diabetic splenocytes, dispersed islet cells from mice on the experimental diet presented a significantly lower in vitro cellular immune aggression. On the other hand, "diabetic" splenocytes from mice fed with the experimental diet produced a significantly reduced cellular immune aggression on dispersed islet cells. Our results showed that feeding branched-chain amino acids increased the capacity of beta cells to withstand a functional assault and diminished the extent of in vitro cellular immune aggression.     

Effector and precursor phenotypes of lymphokine-activated killer cells in mice with severe combined immunodeficiency (scid) and athymic (nude) mice

The lineage of lymphokine-activated killer (LAK) cells is poorly understood. To examine the relationship between LAK and natural killer (NK) cells we utilized two congenitally immunodeficient mice, namely severe combined immunodeficient (scid) and athymic (nude) mice that lack T cells but have normal NK cells. LAK activity was evaluated by the ability to lyze NK-resistant P815 cells. When cultured with human recombinant interleukin 2, splenocytes of scid and nude mice could generate LAK activity at levels comparable to or more than those of normal C.B-17 mice. LAK effector cells in these immunodeficient mice as well as normal mice had the phenotype resembling that of NK cells with asialo-GM1 (aGM1) expression. In vivo treatment with anti-aGM1 antiserum completely abolished the induction of LAK activity from splenocytes of normal mice. In contrast, LAK activity in splenocytes of scid and nude mice was still demonstrable even after this treatment, indicating that most LAK precursors in both mice were cells without aGM1 antigen. The aGM1- progenitors for LAK activity, probably in common with NK progenitors, appeared to be more expanded in scid and nude mice than in normal mice. The use of such congenitally immunodeficient mice should be helpful in studying the differentiation step of LAK as well as NK cells from their precursors.     

Effect of chitin hexamer and thuricin 17 on lignification-related and antioxidative enzymes in Soybean Plants

Inducers of disease resistance in crop plants have a role in sustainable agriculture. We describe a set of bacteriocins that can potentially improve plant growth by controlling specific pathogens and inducing generalized resistance. Solutions of the bacteriocin thuricin 17 and/or a chitin hexamer (a known inducer and positive control) were applied to leaves of two-week-old soybean plants, and levels of lignification-related and antioxidative enzymes were monitored. Phenyl ammonia lyase (PAL) activity in thuricin 17-treated leaves was highest at 60 h after treatment, being 61.8% greater than the control. PAL activity also was increased 18.1% at 72 h after treatment with the chitin hexamer. Tyrosine ammonia lyase (TAL) activity in leaves was 57.0% higher than the control at 48 h after treatment with thuricin 17, while such activity in chitin hexamer-treated leaves was increased by 23.8% at 72 h. At 36 h after treatment with the chitin hexamer or chitin hexamer + thuricin 17, the total concentration of phenolic compounds was 15.3 or 19.3%, respectively, greater than the control. At 72 h, total phenolic concentrations increased by 23.2 and 19%, respectively, in response to thuricin 17 and chitin hexamer + thuricin 17. POD activity in thuricin 17-treated leaves increased by 74.6 and 81.2% at 48 and 72 h, respectively, whereas SOD activity increased by 24.9 and 79.9%, respectively, in chitin hexamer- and thuricin 17-treated leaves at 48 h. A peroxidase isozyme (31 kDa isomer) was induced in thuricin 17-treated leaves at 60 h, while catalase (59 kDa isomer) was induced in chitin hexamer-treated leaves. PAGE showed that two major SOD bands (Fe-SODs) were produced by both types of treatment. Collectively, these results indicate that the bacteriocin thuricin 17 can act as an inducer of plant disease defenses (i.e., activated lignification-related enzymes, antioxidative enzymes, and related isozymes) and that this induction is similar, but not identical, to that of the chitin hexamer elicitor. Although treatment with thuricin 17 + chitin hexamer also induced those responses, it did not present a clear pattern of additivity or synergy.     

Curcumin effects on inflammation and performance recovery following eccentric exercise-induced muscle damage

Downhill running is associated with fiber damage, inflammation, delayed-onset muscle soreness, and various functional deficits. Curcumin, a constituent of the Indian spice turmeric has been investigated for its anti-inflammatory activity and may offset some of the damage and functional deficits associated with downhill running. This study examined the effects of curcumin on inflammation and recovery of running performance following downhill running in mice. Male mice were assigned to downhill placebo (Down-Plac), downhill curcumin (Down-Cur), uphill placebo (Up-Plac), or uphill curcumin (Up-Cur) groups and run on a treadmill at 22 m/min at -14% or +14% grade, for 150 min. At 48 h or 72 h after the up/downhill run, mice (experiment 1) underwent a treadmill performance run to fatigue. Another subset of mice was placed in voluntary activity wheel cages following the up/downhill run (experiment 2) and their voluntary activity (distance, time and peak speed) was recorded. Additional mice (experiment 3) were killed at 24 h and 48 h following the up/downhill run, and the soleus muscle was harvested for analysis of inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), and plasma was collected for creatine kinase analysis. Downhill running decreased both treadmill run time to fatigue (48 h and 72 h) and voluntary activity (24 h) (P < 0.05), and curcumin feedings offset these effects on running performance. Downhill running was also associated with an increase in inflammatory cytokines (24 h and 48 h) and creatine kinase (24 h) (P < 0.05) that were blunted by curcumin feedings. These results support the hypothesis that curcumin can reduce inflammation and offset some of the performance deficits associated with eccentric exercise-induced muscle damage.     

Complementation of the anti-TNP PFC response of N:NIH nude mice from nude dams by spleen cells of nude mice from heterozygous dams.

Type I and Type II nude (nu/nu) mice are borne and raised by nu/nu dams and +/nu dams, respectively, under SPF conditions. Splenocytes from Type I nude mice exhibit even smaller in vitro PFC responses to trinitrophenylated rabbit erythrocytes than splenocytes from Type II nude mice. Type II splenocytes complement the magnitude of the PFC response of Type I splenocytes. The helper cell-like activity contained in Type II splenocyte suspensions was increased by treating Type II nude donor mice with dibutyryl cyclic AMP, but was essentially unaffected by pretreatment of Type II donors with endotoxin (LPS). The helper-like activity of Type II splenocytes was suppressed by treatment with a rabbit antiserum (plus C′) raised against a mixture of neonatal mouse thymocytes (RAM-T). We interpret the data to indicate that Type II splenocytes contain a larger number of residual T cells and/or a more fully developed population of T precursor cells that initiate or that are capable of being induced to initiate a helper function.     

Lymphotoxin-alpha-dependent spleen microenvironment supports the generation of memory B cells and is required for their subsequent antigen-induced activation

Lymphotoxin alpha-deficient (LTalpha-/-) mice show dramatically reduced IgG responses after either primary or secondary immunizations with sheep red blood cells (SRBC). When splenocytes from SRBC-primed wild-type donor mice were infused into irradiated naive wild-type recipient mice, they generated a robust memory IgG response, but not when infused into LTalpha-/- recipients, indicating that the microenvironment that develops in LTalpha-/- mice is incompetent to support the activation of this memory response. When irradiated wild-type mice were reconstituted with splenocytes from primed LTalpha-/- donors and then challenged with the same immunizing Ag, no memory response was observed, indicating further that memory cells could not be generated in the LTalpha-/- environment. To address which lymphocyte subsets were impaired in the LTalpha-/- mice, we performed reconstitution experiments using a hapten/carrier system and T cells and B cells from different primed donors. There was no detectable defect in either the generation or expression of memory T cells from LTalpha-/- donors. In contrast, B cells were not primed for memory in the microenvironment of LTalpha-/- mice. Additionally, primed wild-type memory B cells could not express a memory IgG response in the LTalpha-/- microenvironment. Thus, splenic white pulp structure, which depends on the expression of LTalpha for its development and maintenance, is needed to support the generation of memory B cells and to permit existing memory B cells to express an isotype switched memory Ig response following antigenic challenge.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Repopulation of spleens of irradiated mice after injection of spleen cell subpopulations

Haemopoietic stem cells present in the spleen of adult mice were analysed by grafting X-irradiated animals with polystyrene-nonadherent (NABS) and polystyrene-adherent (ABS) B-enriched splenocytes from syngeneic donors. The progeny of the haemopoietic stem cells present in NABS and ABS subsets were studied with respect to size, surface markers, and response to mitogens and antigens. Ninety-six per cent of the precursors of the myeloid cell lineage (CFU-S) were present in the NABS fraction (50-fold enrichment). The presence in NABS of progenitors of functional T and B lymphocytes was also demonstrated. Twelve days after grafting with NABS, more than 80% of the recipient splenocytes were large and nonadherent granulocyte-like cells. These cells had surface similarities with NABS from normal mice, since both populations reacted with peanut agglutinin and with a rabbit anti-NABS (RAN) serum.