Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

山萘酚通过增强Treg细胞免疫抑制功能延长移植物生存时间

目的:探讨应用山萘酚增强Treg细胞免疫抑制功能,从而抑制大鼠移植物排斥反应并改善移植物生存的作用和机制。方法:以Wister大鼠和SD大鼠分别为供、受体,建立同种异体皮肤移植排斥反应动物模型。观察受体老鼠皮肤移植物的情况,记录移植物失功时间(移植物皮片80%面积发生排斥)。RT-PCR检测移植7天后脾细胞、淋巴细胞FOXP3、CTLA-4和IL-10的mRNA水平,用HE染色组织病理学观察术后7天移植皮片的淋巴细胞浸润程度。体外实验T细胞增殖抑制试验加入山萘酚作为对照,观察Treg功能情况。结果:1.山萘酚能增强移植后同种异体移植物的生存时间(DMSO组6.3±0.3天,山萘酚组13.7±0.39天,P<0.01);2.RT-PCR显示山萘酚可增强细胞CTLA-4(对照组9.24±0.17,山萘酚组12.48±0.145,P<0.05)、FOXP3(对照组0.96±0.07,山萘酚组1.41±0.07,P<0.01)和IL-10(对照组0.95±0.12,山萘酚组1.50±0.16,P<0.05)的mRNA水平;3.体外T细胞增殖抑制实验中,山萘酚可增强Treg细胞的免疫抑制功能。结论:在大鼠皮肤移植模型中,山萘酚可延长皮肤移植物的生存时间,提高Treg细胞相关IL-10、FOXP3和CTLA-4的mRNA水平;体外实验中,能抑制效应T细胞的增殖,表明山萘酚在提高移植物生存方面存在一定的价值。     

Effects of bone marrow-derived cells on monocrotaline- and hypoxia-induced pulmonary hypertension in mice

Background

Bone marrow -derived cells (BMDCs) can either limit or contribute to the process of pulmonary vascular remodeling. Whether the difference in their effects depends on the mechanism of pulmonary hypertension (PH) remains unknown.

Objectives

We investigated the effect of BMDCs on PH induced in mice by either monocrotaline or exposure to chronic hypoxia.

Methods

Intravenous administration of the active monocrotaline metabolite (monocrotaline pyrrole, MCTp) to C57BL/6 mice induced PH within 15 days, due to remodeling of small distal vessels. Three days after the MCTp injection, the mice were injected with BMDCs harvested from femurs and tibias of donor mice treated with 5-fluorouracil (3.5 mg IP/animal) to deplete mature cells and to allow proliferation of progenitor cells.

Results

BMDCs significantly attenuated PH as assessed by reductions in right ventricular systolic pressure (20 ± 1 mmHg vs. 27 ± 1 mmHg, P ≤ 0.01), right ventricle weight/left ventricle+septum weight ratio (0.29 ± 0.02 vs. 0.36 ± 0.01, P ≤ 0.03), and percentage of muscularized vessels (26.4% vs. 33.5%, P ≤ 0.05), compared to control animals treated with irradiated BMDCs. Tracking cells from constitutive GFP-expressing male donor mice with anti-GFP antibodies or chromosome Y level measurement by quantitative real-time PCR showed BMDCs in the lung. In contrast, chronically hypoxic mice subjected to the same procedure failed to show improvement in PH.

Conclusion

These results show that BMDCs limit pulmonary vascular remodeling induced by vascular injury but not by hypoxia.     

Effects of the chromatographic fractions of the pig placental trophoblast on graft-versus-host reaction

The trophoblast has a significant role in regulation of immune reactions at the materno-fetal interface by producing biologically active substances. In our previous studies five fractions with immunomodulatory activities were isolated by gel chromatography from trophoblast of pig placentas. To confirm the immunomodulatory effect of these trophoblast fractions on allogeneic in vivo systems and to obtain more evidence for the relevance of their activity on the maternofetal interface, their effect was studied on graft-versus-host reaction (GVHR). To assess the GVHR, the primary and secondary popliteal lymph nodes assay was used in mice. In the primary GVHR, 100 microg protein of Fraction 2-5, mixed with 5 x 10(6) allogeneic spleen cells (C57BL/6), were injected into one of the foot pads of recipient (BALB/c) mice. The secondary GVHR was induced in F1 (BALB/c x C57BL/6) mice by injection of spleen cells of BALB/c mice intraperitoneally preimmunized with allogeneic cells. The GVHR was measured by the weight of lymph nodes and by the lymphocyte proliferation. Flow cytometric analyses of the cells in the nodes with GVHR and under the influence of Fraction 4 or 5 were performed using monoclonal antibodies. In the primary GVHR, Fraction 4 or 5, injected simultaneously with allogeneic spleen cells, significantly suppressed the lymph nodes reactivity. Fractions 4 and 5 inhibited the ability of the spleen cells of mice intraperitoneally preimmunized with allogeneic cells to induce secondary GVHR in F1 mice. The Fraction 2 and 3 had no effect on GVHR. The results revealed that a group of proteins with Mr 37-7 kDa, isolated from trophoblast of pig placenta, strongly suppressed popliteal lymph node reactivity in the primary and secondary GVHR. The data provide convincing evidence for these fractions in vivo activity, for their effect across the species barrier and suggest the relevance of the same reactions on the materno-fetal interface.     

Immunomodulation property of hexane fraction of leaves of Cinnamomum tamala Linn. in rats

The leaves of Cinnamomum tamala Linn. (CT) (Lauraceae) clinically used in Ayurveda as antidiabetic and diuretic, but no reports are available towards immunomodulating property. Its hexane fraction (CTH) was orally given to rats for 10 days and delayed type of hypersensitivity (DTH), antibody producton against sheep red blood cells (SRBCs), mitotic index in bone marrow cells and concanavalin A (Con A) mediated proliferation of lymphocytes were assessed. Further on 30 days treatment, change in body weight (BW), spleen weight, thymus weight, bone marrow cellularity and hematological changes were observed. It inhibited significantly the DTH response (IC₅₀ 1475 ± 57.19 mg kg^?1 BW), antibody production, suppressed mitotic index in bone marrow cells along with the suppression of lymphocyte proliferation against Con A (IC₅₀ 63.33 ± 1.95 µg mL^?1). In all experiments, cyclophasphamide and dexamethasone had been used as reference drug for in vivo and in vitro studies, respectively. On 30 days treatment, the CTH (800 mg kg^?1 BW and above) significantly suppressed growth rate, increase of spleen and thymus weight and low bone marrow cellularity. In hematological examination, it inhibited total white blood cell and lymphocytes count and increased per cent of polymorphs. Thus, it could be suggested that the fraction possesses immunosuppressive property at doses, higher than 800 mg kg^?1 BW in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Synergistic anti-suppressor effect of mini cells prepared from Salmonella typhimurium and mitomycin C in EL 4-bearing mice

Summary The subcutaneous growth of EL4 cells was significantly accelerated when they were injected together with spleen cells collected from mice which had received EL4 cells SC 14 days previously, and all mice died within 18 days after receiving this mixture; 80% of mice which received a mixture of EL4 and spleen cells collected immediately after EL4 graft survived over 40 days. Spleen cells collected 14 days after EL4 graft suppressed the blastogenic responses of normal spleen lymphocytes to concanavalin A, pokeweed mitogen, and lipopolysaccharide of Escherichia coli in a mixed culture system.Acceleration of tumor growth was retarded when EL4 cells were injected together with spleen cells from EL4-bearing mice treated with both Salmonella typhimurium mini-cells and mitomycin C, and 60% of mice survived over 40 days. Blastogenic responses of normal spleen lymphocytes mixed with spleen cells from EL4-bearing mice treated with both mini-cells and mitomcycin C were restored almost to control levels. The results suggest that combination treatment with mini-cells and mitomycin C synergistically inhibits the induction of suppressor cells in EL4-bearing mice.     

葛根素对U14宫颈癌小鼠血液流变学、脾淋巴细胞增殖及细胞毒性影响

摘要 目的：研究葛根素治疗对U14宫颈癌小鼠血液流变学、脾淋巴细胞增殖活性及对宫颈癌细胞毒性的影响。方法：45只雌性昆明小鼠随机分为对照组、模型组和葛根素组。模型组和葛根素组小鼠通过腋下注射U14小鼠宫颈癌细胞建立U14宫颈癌移植瘤小鼠,并且葛根素小鼠通过葛根素灌胃进行治疗,对照组和模型组小鼠给予等量生理盐水。比较各组小鼠血流变学、脾淋巴细胞增殖活性及对宫颈癌细胞毒性。结果：经葛根素治疗的葛根素组宫颈癌小鼠肿瘤重量显著低于模型组小鼠（P<0.05）,葛根素治疗宫颈癌小鼠的抑瘤率是（42.91±12.91）%。宫颈癌小鼠低切/高切全血粘度、血浆粘度值以及血细胞比容均显著升高（P<0.05）,而葛根素治疗可显著降低宫颈癌小鼠低切/高切全血粘度、血浆粘度值以及血细胞比容（P<0.05）。宫颈癌小鼠脾脏重量、脾脏指数和脾淋巴细胞体外增殖能力均显著下降（P<0.05）,而葛根素治疗可显著提高宫颈癌小鼠脾脏重量、脾脏指数和脾淋巴细胞体外增殖能力（P<0.05）。此外,经葛根素治疗的宫颈癌小鼠脾淋巴细胞对U14宫颈癌细胞细胞毒性显著高于模型组宫颈癌小鼠（P<0.05）。结论：葛根素治疗可降低U14宫颈癌小鼠血液粘度、改善血流变性质,并且可以提高脾淋巴细胞的增殖活性和对宫颈癌细胞的杀伤力。     

Properties of reticulum cell sarcomas in SJL/J mice. III. Promotion of tumor growth in irradiated mice by normal lymphoid cells.

Experiments were performed to elucidate the mechanisms by which lymphocytes obtained from an M-antigen-incompatible strain reduce the specific mixed lymphocyte culture (MLC) response of lymphoid cell populations after injection into allogeneic recipients. Mice of strain CBA were injected with spleen cells from hybrids of the H-2-compatible, M-antigen-incompatible strain C3H. Normal C3H × CBA spleen cells increased the MLC reactivity of the host's lymphocytes during the first 1–3 days, and thereafter the response against C3H was drastically reduced. Mitomycin-treated or antibody-coated C3H × CBA cells rather enhanced the MLC responsiveness. Roughly similar results were obtained by injecting untreated H-2-incompatible C3H hybrid lymphocytes. Lymph node or spleen cell populations from CBA mice, injected with C3H × CBA cells up to 2 weeks earlier, were found to depress the MLC reactivity against C3H when transferred to new CBA hosts. The results indicate that injected cells had survived for 2 weeks in the host. On the other hand, H-2-incompatible C3H hybrid cells could not be detected even at day 3 after injection into CBA mice. The results also indicate that C3H hybrid lymphocytes have to be functionally intact and able to survive in the host for a relatively long period of time to be able to reduce the specific MLC response of the host's lymphocytes.     

The importance of experience in the development of tolerance to ethanol hypothermia

Studies were conducted to determine if an animal has to experience a reduction in body temperature during the acquisition period in order to develop tolerance to the hypothermic effect of ethanol. Adult, drug-naive C57BL/6J mice were injected with 2.6 or 3.6 g/kg ethanol or normal saline once daily for 6 days. During the tolerance acquisition period, days 1–5, mice were placed into warmed chambers (36 ± 2°C) which offset ethanol hypothermia or into chambers at room temperature (24 ± 1°C). On day 6, all mice were injected with ethanol and placed into chambers at room temperature. Tolerance to ethanol's hypothermic effect did not develop in the ethanol-warm acquisition group. These mice had a significantly greater degree of hypothermia on test day than the ethanol-room temperature acquisition group, which showed tolerance, and their degree of hypothermic response was similar to that of mice injected with saline during acquisition. The differences between groups cannot be attributed to pharmacokinetic alterations or to conditioned responses since there were no differences between groups in blood or brain ethanol concentrations on test day and all groups were exposed to the same acquisition and test situations. These results extend previous work to suggest that the development of tolerance to the physiological, as well as behavioral, aspects of ethanol intoxication requires more than simple exposure to ethanol.     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。     

KINETICS OF ERYTHROPOIETIN STIMULATED PROLIFERATION OF ERYTHROID PROGENITORS IN THE SPLEEN

The effect of RBC transfusion and erythropoietin (EPO) on the proliferation of immature erythrocyte progenitors was studied in the spleens of RBC transfused, lethally irradiated mice injected with bone marrow. Transfusion decreased expansion of the progenitors and slowed their proliferation: the mean cycle time as measured by per cent labelled mitosis (PLM) on the third day after injection of bone marrow was 10.7 hr in transfused as compared to 5.6 hr in non-transfused mice. One injection of five units of erythropoietin on day 2 decreased the mean cycle time to 7.3 hr in transfused mice and increased expansion of the progenitor cells. The effects of erythropoietin on cell proliferation were prompt: a significant increase of incorporation of ³H-TdR into DNA occurred within 2 hr of injection. Erythroblasts were absent from the spleens of transfused, irradiated bone marrow injected mice; however, erythroblasts appeared by 72 hr and 48 hr following EPO injection either 2 days or 5 days after transplantation respectively. Increased uptake of radioactive iron in spleen after erythropoietin injection preceded the appearance of erythroblasts by 2 and 1 days when erythropoietin was injected either 2 or 5 days after marrow transplantation respectively. The increase in cellular proliferation induced by erythropoietin in transfused irradiated mice injected with bone marrow equivalent to 0.35 femoral shaft was manifested as an increase of the total DNA content in the spleen by 119 μg (11.9 × 10⁶ cells) within 48 hr of injection. The cellular increment produced by EPO injection on day 5 to mice given 0.05 femoral shaft consisted mainly of undifferentiated mononuclear cells, most of which were labelled, with erythroblasts comprising only one quarter of the increment. Erythropoietin inactivated by mild acid hydrolysis failed to increase cellular proliferation.     

Intravascular and extracellular volumes in the diabetic rat

Simultaneously measured intravascular (IVV) and extracellular (ECV) volumes in diabetic rats have not been reported. We evaluated IVV and ECV in alloxan induced diabetic rats which were either untreated (DU) or received supplemental daily insulin (DI) for three months. Two separate groups of control rats were comparably weight matched to each experimental group. Radio-iodinated (125-I) human serum albumin (RISA) and 35-S sulfate were used to determine IVV and ECV respectively. In DU rats, values for IVV and ECV expressed as a percentage of body weight were 9.3±0.5% and 35±2% respectively; both are significantly larger than the volumes measured in control rats (IVV=6.6±0.2%, p^<0.001 and ECV=28±1%, p<0.01). DI rats had volumes (IVV=6.0±0.3% and ECV=24±3%) which were not significantly different than those of control rats (IVV=5.7±0.1% and ECV=22±1%). Thus, untreated diabetic rats had increased IVV and ECV while diabetic rats that received insulin were normovolemic despite the presence of hyperglycemia.     

Cytogenetic studies on the effect of feeding mice with stored wheat grains treated with malathion

The cytogenetic effect of malathion residues in wheat grains stored for different periods of time (4, 12, 24 weeks) was evaluated in Swiss mice. The studies included: (1) chromosomal aberrations analysis in bone-marrow and spermatocyte cells; (2) chromosomal aberrations and sister chromatid exchange (SCE) analysis in spleen cell culture from mice fed with stored wheat grains. The tested doses were 8.36 (applied dose), 25.08 and 41.80 mg malathion kg⁻¹ wheat grains. The results demonstrated that the cytogenetic effect induced in different mouse tissues by malathion residues was dose-dependent and increased with increasing of both feeding and storage periods.Feeding mice with wheat grains stored for 4 weeks had a non-significant effect with respect to the induction of chromosomal aberrations or SCEs. Significant chromosome damage and increase of SCEs were observed in mice fed with wheat grains stored for 12 weeks. The maximum effect was recorded in mice fed for 12 weeks with the grains treated with the highest tested dose and stored for 24 weeks. However, mitomycin C i.p.-injected in mice at 1 mg kg⁻¹ body weight (b.w.) (positive control) induced a higher effect. The percentage of chromosome aberrations reached 13.60±0.98, 13.60±0.77 and 11.73±0.98 (P<0.01) in bone-marrow, cultured spleen cells and spermatocytes, respectively. The significant increase of abnormalities in spermatocytes was seen for univalent formation only, predominantly of the sex chromosomes. The frequency of SCEs was 10.76±0.62 per cell (P<0.01) in cultured spleen cells compared with 5.46±0.45 per cell for control and 14.66±0.54 per cell for the positive control.The obtained results indicate that malathion residues in stored wheat grains have potential genotoxic effect in mice under the conditions tested.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

Entamoeba histolytica: antibody responses and lymphoreticular changes in gerbils (Meriones unguiculatus) in response to experimental liver abscess and amebic extract infection

Antibody responses and histological changes in hepatic lymph nodes and spleen of gerbils (Meriones unguiculatus) during the course of experimental hepatic amebiasis (5-60 days), or in those injected with extracts of Entamoeba histolytica, are described. Lymph node and spleen responses in infected animals paralleled the proliferation of the amebic liver abscess. However, spleen follicle responses were similar in animals that received low or high doses of the amebic extract and differed histologically from those with amebic liver abscess. Liver abscesses, up to 30 days postinfection (pi), doubled in weight between 10 and 15 and between 20 and 30 days pi. Early changes (10 days pi) in the lymphoreticular tissues were characterized by increased size and weight of the organs, hyperplastic follicles, and blastogenesis in the T-dependent areas. At 20 and 30 days pi, the size of spleen follicles increased and there was depletion of lymphocytes from the periarterial area (PAA), as well as gross extension of the red pulp, accompanied by extramedullary erythropoiesis and megakaryocytosis. The paracortical areas (PCA) of lymph nodes were depleted of lymphocytes and histiocytosis throughout the organ, and there was intense plasma cell activity in the medulla. At 60 days pi, lymphocyte repopulation was noted in the PCA and PAA; germinal centers were depleted of blast cells and the spleen red pulp had contracted. Antiamebic antibody titers were low throughout the infection. Changes in the cellularity of the lymphoid organs are discussed in relation to the proliferation of the amebic liver abscesses in infected animals and in those which were injected with the amebic extract.     

In vivo delivery of interleukin-6 using vaccinia virus: effects on T lymphocytes in nude mice

We have constructed a recombinant vaccinia virus (VV) expressing the human interleukin-6 (IL-6) gene, VV(IL-6). After injection of VV(IL-6) i.v. into Balb/c mice, circulating IL-6 was detected during 3 days with the peak activity on day 4, indicating that VV injection is an effective method to deliver lymphokines in vivo. We have further examined the effects of IL-6 in vivo in immunodeficient mice. Nude mice were injected i.v. with VV(IL-6). Ten days after the injection, mice were sacrificed and spleen cells were obtained. Spleen cells from VV(IL-6) injected mice proliferated remarkably in response to IL-2, while spleen cells from mice injected with unrelated VV manifested no particular proliferation in response to lymphokines. When spleen cells were further cultured in vitro for 5 days in the presence of Concanavalin-A stimulated rat spleen cell supernatant (Con-A factor), CD4 or CD8 positive cells were detected in the VV (IL-6) injected group, while few positive cells were detected in the control groups. These results suggest that IL-6 stimulates nude mice spleen cells in vivo, to a stage where they are able to proliferate in response to IL-2, or to differentiate into CD4 or CD8 positive cells in presence of rat Con-A factor.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.