Immunoendocrine modulation and stimulation of hematopoiesis with the ionophore copolymer T150R1

T150R1, an 8000-dalton copolymer with sodium ionophore activity, has been shown to modulate cellular responses in multiple systems. In this article, we studied its effects on lymphoid and hematopoietic organs in the context of the adrenal-pituitary axis. When injected in mice as an oil in water emulsion, T150R1 caused a rapid, profound, and dose-dependent thymic involution accompanied by splenic hyperplasia. Time course experiments with a 2.5-mg dose revealed that the thymus size was minimal at Day 2, rose to normal by Day 14, then enlarged and gradually returned to normal by Week 6 postinjection. Thymic involution was due to cellular depletion of the cortical area, whereas thymic enlargement was due to cortical hyperplasia. Splenomegaly was seen as early as Day 4, peaked by Day 14, and gradually returned to normal by Week 6. The splenic enlargement was due to hyperplasia of the red pulp, with evidence of proliferating erythropoietic, myelopoietic, and megakaryopoietic precursors. In addition, the bone marrow was stimulated and extramedullary hematopoiesis was present in the liver. The effects of T150R1 on the thymus appeared to be mediated by corticosteroids while the effects on hematopoiesis were not. Corticosterone and ACTH levels were increased in treated animals. Adrenalectomy diminished the T150R1-induced thymic involution but enhanced the splenic hyperplasia. Hypophysectomy did not prevent thymic involution, suggesting that T150R1 has endocrine stimulatory effects. These data suggest that T150R1 represents a new class of ionophores which may act on excitable cells within the endocrine, immune, and hematopoietic systems.     

The immunoproliferative response of mice to intravenously or subcutaneously injected BCG

Summary Intravenous injection of BCG caused (1) a transient thymic epithelial hyperplasia with increase of PAS-positive cells in the cortex and medulla which showed the pronounced secretory activity of a substance which could be histochemically identified as an acid mucopolysaccharide; (2) an equally transient increase in the number of pyroninophilic lymphocytes with increased polyribosome content of the cells and mitoses in the thymic cortex; this reached a peak on day 6 following the injection but was unassociated with an increase in thymic weight; and (3) a systemic granulomatous histiocytic reaction in the liver, spleen, lungs, and lymph nodes, but not in the thymus, bone marrow, or Peyer's patches. The significance of the thymic epithelial changes is not clear but it did coincide with increased pyroninophilia and mitotic activity of the thymic cortical cells, suggesting a possible interaction between this secretory product and the thymic cortex. Comparing the thymic changes with the thymus of other animals of the same species injected with i.v. or i.p. LPS, i.v. or s.c. HIU II fraction of BCG, i.v. pertussis vaccine, i.p. complete or incomplete Freund's adjuvant, and killed at the same planned intervals after the injection of the adjuvants, BCG proved to have a unique action on the thymus with regard to both lymphocytic and epithelial changes. Hepatic, pulmonary, and splenic histiocytic granulomas were observed only in those animals injected intravenously with BCG.     

Lymphoid organ development in Xenopus thymectomized at eight days of age

This paper examines the effect of early thymectomy on the subsequent development of lymphoid tissues in the toad, Xenopus laevis. At the time of thymic removal (8 days post-fertilization) all the lymphoid organ anlagen are at a rudimentary state of differentiation and contain few, if any, small lymphocytes. Despite the absence of any thymic tissue all thymectomized animals grew normally. Thymectomized larvae developed relatively normal lymphoid organs. However, lymphoid depletion was apparent in the splenic red pulp and in the pharyngeal ventral cavity bodies. Examination of the lymphoid organs of post-metamorphic Xenopus revealed reduction in spleen size following thymectomy. Lymphoid depletion was evident in the splenic red pulp of many thymectomized toadlets and reduction in proportion of white to red pulp was also noted in a few of these animals. Absence of the thymus had no apparent effect on the histology of the other lymphoid organs examined.     

Age-related hyperplasia of the thymus and T-cell system in the Buffalo rat

This report describes the development of hyperplasia of both the thymus and the peripheral T-cell system with advancing age in the Buffalo rat. Buffalo/Mna rats do not show age-related thymic involution, but rather develop thymic hyperplasia with advancing age. This thymic growth is expansile and there is no infiltration of the surrounding tissues. Because the enlarging thymus occupies the thoracic cavity, most of the rats die of respiratory failure by the age of 24 months. Thymic enlargement is due to primary hyperplasia of cortical epithelial cells and the large number of proliferating lymphocytes. The hyperplastic epithelial cells are bizarre in shape and strongly positive when stained with Th-3 monoclonal antibody (MoAb), anti-thymosin antibody and anti-EGF antibody, but negative with Th-4 MoAb. The patterns of distribution of CD-5+, CD-4+ and CD-8+ lymphocytes within the hyperplastic thymus are similar to those seen in young rats of other species. The high level of T-cell emigration from the thymus to the periphery appears to persist throughout life, since the percentage of normal splenic T-cells also increase with advancing age and exceed 70% of the total by 24 months of age. This thymic enlargement with abnormal hyperplasia of cortical epithelial cells can be prevented by hypophysectomy.     

Expression of insulin-like growth factor II (IGF-II) and histological changes in the thymus and spleen of transgenic mice overexpressing IGF-II

 Previously, transgenic mice were constructed overexpressing human insulin-like growth factor II (IGF-II) under control of the H₂k^b promoter. The IGF-II transgene was highly expressed in thymus and spleen, and these organs showed an increase in weight. In the current study we have analyzed the sites of IGF-II mRNA expression, the distribution of IGF-II, IGF-I, and both IGF receptors, and histomorphometrical changes in thymus and spleen. With in situ mRNA hybridization, expression of the IGF-II transgene is found with high intensity in the thymic medulla and in the white pulp/marginal zone of the spleen, whereas there were scattered positive cells in the thymic cortex and in the splenic red pulp. Hybridization was restricted to non-lymphocytic cells. Immunohistochemistry revealed intense IGF-II peptide staining with the same distribution as IGF-II mRNA. There was additional intense IGF-II staining of all elements in the splenic red pulp (including trabeculae) and diffuse, low level staining in the thymic cortex. These findings were not observed in control mice. In the thymic medulla, most IGF-II producing cells co-labelled with keratin, whereas a minor population also stained for the monocyte/macrophage marker MOMA-2. In the spleen, co-labelling of IGF-II producing cells was found with MOMA-1 (marginal zone), or with the dendritic cell marker NLDC-145 (red pulp). IGF-I and both IGF receptors were found in these organs in nearly all cell types, with a similar pattern in transgenic mice and in control animals. Histomorphometric analysis revealed a marked increase of thymus cortex size and an increased trabecular size in the spleen. This suggests that IGF-II overproduction induces local effects (auto/paracrine) in the thymic cortex, but not in the thymic medulla. Trabecular growth in the spleen most likely is a distant effect (paracrine or endocrine) of IGF-II overproduction. Accepted: 5 September 1996     

巨细胞病毒感染对新生豚鼠胸腺的影响

新生豚鼠皮下接种豚鼠巨细胞病毒(GPCMV)后,导致动物胸腺急性感染。感染豚鼠胸腺在接种后第五天开始出现病毒,第十天达高峰。此外,感染动物胸腺的发育受到抑制,细胞总数和T淋巴细胞数随朐腺中病毒滴度的增高而进行性下降,至接种GPCMV后第十天最显著。由于病毒对T细胞的作用,细胞表面红细胞受体的丧失导致胸腺Null细胞百分比高于对照动物。     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

Effect of histamine on the rosette-forming capacity of T-lymphocytes in immunization with ADPT vaccine and its components

The results obtained in the study of the influence of histamine on the capacity of T-lymphocytes of guinea pigs immunized with DPT-vaccine and its components for spontaneous rosette formation are presented. Histamine at a concentration of 10(-3) M has been found to inhibit the capacity of blood and splenic lymphocytes of guinea pigs immunized with adsorbed DPT vaccine for spontaneous rosette formation. The inhibitory effect is more pronounced after the immunization of the animals with adsorbed DPT vaccine and Bordetella pertussis suspension.     

急性巨细胞病毒感染导致新生豚鼠脾细胞亚群的定量改变

新生豚鼠皮下接种豚鼠巨细胞病毒(GPCMV)后,导致脾脏GPCMV急性感染,脾脏肿大,脾细胞增生,计算表明,脾T淋巴细胞,B淋巴细胞、吞噬细胞数显著高于正常动物,接种病毒后第5天即可在脾细胞检出高滴度感染性病毒(达2.5 log10 TCID50/10~7细胞),其中,主要是脾B淋巴细胞、T淋巴细胞和巨噬细胞携带病毒。     

Activation of lymphoid cells by BCG in vitro

Bacillus Calmette-Guerin (BCG) stimulated splenic and thymic lymphocytes in vitro as measured by uptake of ³H-thymidine. This activation of lymphocytes by BCG required the presence of a critical concentration of macrophages. Thymus cells containing no more than 0.25% macrophages were stimulated by BCG, but reduction of macrophages below this level by adherence to plastic abolished the response. Reconstitution with purified macrophages completely restored the response. A high concentration of adherent cells (“macrophages”) depressed the response of splenic lymphocytes, as judged by the improvement in DNA synthesis after reduction of the proportion of adherent cells in the spleen cell population.Bacillus Calmette-Guerin augmented the production of lymphocyte-activating factor (LAF) from purified splenic adherent cells, but the presence of lymphocytes made that augmentation considerably greater.These data reaffirm the bidirectional nature of the relationship between lymphocytes and macrophages. They further show that BCG can create highly activated populations of each type of cell, in part by enhancing their interaction.     

Effects of a protein-free diet on the DNA-synthesizing and dividing cells in the lymphoid organs of rats. Changes induced by phytohemagglutinin and cortisone invivo

Levels of ³H-thymidine-labeled lymphocytes on autoradiographs of cell smears and mitotic indices (M.I.) were determined in the popliteal lymph nodes (PLN), spleen and thymus of rats fed with a balanced or a protein deprived (PD) diet for 7 weeks. The latter diet reduced the number of labeled cells per 10³ in the spleen but not in the PLN's and the thymus. The M.I.'s were reduced 50% in the spleen and dropped drastically in the thymus. Subplantar injection of phytohemagglutinin (PHA) provoked a sharp increase in the M.I.'s of the PLN's and the spleen, and also moderately increased the labeling index in the PLN's in normal rats. In PD rats PHA no longer influenced the mitoses in the PLN's and the spleen while it considerably increased the proportion of labeled cells in the latter. These discrepancies between DNA-synthesis and mitoses in the thymus and the spleen suggest a premitotic block of the cell cycle after protein deprivation. The effects of a 5 day-cortisone treatment resembled in a large measure the changes induced by a protein-free diet including the arrest of the cell cycle. The thymic lymphocytes were more sensitive to the hormone in PD than in normal rats. On the contrary, in the PLN's and the spleen mainly cortisone-resistant cells remained alive among the DNA-synthesizing lymphocytes after protein deprivation. All these data agree with the hypothesis of an intervention of endogenous glucocorticoids in the protein deficiency-induced lymphoid involution.     

Human, guinea pig and rat lympocyte rosette formation with spermatozoids]

In the thymus, spleen and bone marrow of adult guinea pigs, 14--30-week human fetuses, and the peripheral blood of sterile men there were found cells capable of forming the rosettes with homo- or heterologous spermatozoa (RFC). Development of autoimmune orchitis after the trauma of the rat testis or after the guinea pig immunization with the testicular homogenate mixed with complete Freund's adjuvant caused the appearance of RFC with spermatozoa in the thymus and the spleen of rats, and an increase of their number in the lymphoid organs of guinea pigs. Such treatment did not influence the quantity of sheep-cell rosettes in the lymphoid organs of rats and guinea pigs. A possibility of using the detected capacity of animal and human lymphocytes to form spontaneous and immune rosettes with the spermatozoa to test the degree of lympocyte differentiation and their sensitisation to the spermatozoa antigens after the spermatogenesis distrubances of the autoimmune nature is discussed.     

Sites of action of soltriol (vitamin D) in hamster spleen,thymus, and lymph node,studied by autoradiography

Summary Sibirian hamsters (Photopus sungorus) were injected with³H dihydroxycholecalciferol (vitamin D, soltriol). Autoradiograms of spleen, thymus, and lymph nodes revealed nuclear concentration of the hormone in a select population of cells in all of these organs. In the spleen, labeled cells were abundant in the red pulp, but sparse in the white pulp. In the periarterial lymphatic sheath (PALS) labeled cells were found predominatly at the outer rim, with a few scattered labeled cells in the inner PALS and in the marginal zone. Lymphocytes, including pyronin-positive plasma cells, did not display nuclear labeling. In the red pulp, some of the labeled cells contained pigmented inclusions in the cytoplsm, while most of the labeled cells did not appear phagocytic under the conditions of the experiment. In the thymus, labeled cells were most numerous in the medulla, but sparse in the cortex. Many of the thymic target cells were larger than the unlabeled lymphocytes, with a large and pale nucleus, sometimes containing a distinct nucleolus, and with large and dendritic cytoplasm, having the appearance and distribution of epithelio-reticular cells. In lymph nodes, scattered labeled cells were conspicuous in or near the subcapsular sinus, while other cells did not concentrate radioactivity in their nuclei. The results indicate that nuclear receptors and direct genomic actions for soltriol exist in certain cell populations of lymphatic tissues that probably include reticular cells and a subpopulation of macrophages. These target cells may mediate effects of the steroid on lymphocytes that appear to have no or only very low numbers of nuclear receptors.     

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals.When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.     

Thymus-derived lymphocyte differentiation and lymphocytic leukemias. I. Evidence for the existence of functionally different subpopulations of thymus-derived cells in leukemic AKR mice

The effects of transplanting thymic (LTC), splenic (LSC), and lymph node (LLNC) lymphocytes derived from overtly leukemic AKR mice into preleukemic syngeneic animals were studied. Each of these thymus-derived (T cell) populations produced a different and distinct pathology in recipient mice. Animals receiving LTC exhibited thymoma and enlargement of peripheral lymphoid tissues. Gross organomegaly was also noted in mice given LSC, but thymic atrophy was uniformly observed. The thymus appeared normal in mice receiving LLNC, but marked enlargement of peripheral lymphoid tissues again were observed. The differences noted in disease pathologies correlated with the “homing” patterns of the subpopulations investigated. These findings suggest that subpopulations of T cells exist in mice with a thymus-derived neoplastic disorder.     

Sites of action of soltriol (vitamin D) in hamster spleen, thymus, and lymph node, studied by autoradiography

Siberian hamsters (Photopus sungorus) were injected with 3H dihydroxycholecalciferol (vitamin D, soltriol). Autoradiograms of spleen, thymus, and lymph nodes revealed nuclear concentration of the hormone in a select population of cells in all of these organs. In the spleen, labeled cells were abundant in the red pulp, but sparse in the white pulp. In the periarterial lymphatic sheath (PALS) labeled cells were found predominantly at the outer rim, with a few scattered labeled cells in the inner PALS and in the marginal zone. Lymphocytes, including pyronin-positive plasma cells, did not display nuclear labeling. In the red pulp, some of the labeled cells contained pigmented inclusions in the cytoplasm, while most of the labeled cells did not appear phagocytic under the conditions of the experiment. In the thymus, labeled cells were most numerous in the medulla, but sparse in the cortex. Many of the thymic target cells were larger than the unlabeled lymphocytes, with a large and pale nucleus, sometimes containing a distinct nucleous, and with large and dendritic cytoplasm, having the appearance and distribution of epithelio-reticular cells. In lymph nodes, scattered labeled cells were conspicuous in or near the subcapsular sinus, while other cells did not concentrate radioactivity in their nuclei. The results indicate that nuclear receptors and direct genomic actions for soltriol exist in certain cell populations of lymphatic tissues that probably include reticular cells and a subpopulation of macrophages. These target cells may mediate effects of the steroid on lymphocytes that appear to have no or only very low numbers of nuclear receptors.     

Intranasal Mucosal Boosting with an Adenovirus-Vectored Vaccine Markedly Enhances the Protection of BCG-Primed Guinea Pigs against Pulmonary Tuberculosis

Background

Recombinant adenovirus-vectored (Ad) tuberculosis (TB) vaccine platform has demonstrated great potential to be used either as a stand-alone or a boost vaccine in murine models. However, Ad TB vaccine remains to be evaluated in a more relevant and sensitive guinea pig model of pulmonary TB. Many vaccine candidates shown to be effective in murine models have subsequently failed to pass the test in guinea pig models.

Methods and Findings

Specific pathogen-free guinea pigs were immunized with BCG, AdAg85A intranasally (i.n), AdAg85A intramuscularly (i.m), BCG boosted with AdAg85A i.n, BCG boosted with AdAg85A i.m, or treated only with saline. The animals were then infected by a low-dose aerosol of M. tuberculosis (M.tb). At the specified times, the animals were sacrificed and the levels of infection in the lung and spleen were assessed. In separate studies, the long-term disease outcome of infected animals was monitored until the termination of this study. Immunization with Ad vaccine alone had minimal beneficial effects. Immunization with BCG alone and BCG prime-Ad vaccine boost regimens significantly reduced the level of M.tb infection in the tissues to a similar extent. However, while BCG alone prolonged the survival of infected guinea pigs, the majority of BCG-immunized animals succumbed by 53 weeks post-M.tb challenge. In contrast, intranasal or intramuscular Ad vaccine boosting of BCG-primed animals markedly improved the survival rate with 60% of BCG/Ad i.n- and 40% of BCG/Ad i.m-immunized guinea pigs still surviving by 74 weeks post-aerosol challenge.

Conclusions

Boosting, particularly via the intranasal mucosal route, with AdAg85A vaccine is able to significantly enhance the long-term survival of BCG-primed guinea pigs following pulmonary M.tb challenge. Our results thus support further evaluation of this viral-vectored TB vaccine in clinical trials.     

Amino acid transport in thymic- and spleen-derived lymphocytes.

We have examined the transport of amino acids by the sodium-dependent "A" and "ASC" system in thymic- and splenic-derived lymphocytes from the Long-Evans rat. Lymphocytes derived from the thymus transport amino acids by both the "A" and "ASC" systems, whereas lymphocytes from the spleen transport amino acids by the "ASC" system only. Thymic lymphocytes are capable of establishing a steady state distribution ratio of 7.9 for 2-aminoisobutyric acid, but splenic lymphocytes can attain only 3.5. The steady state distribution ratio of alanine was the same in both cell types. Sodium-independent transport is also different in splenic and thymic lymphocytes. But both cells move amino acids by a Na+-independent system for mediated exchange-diffusion. The studies show that lymphocytes derived from the spleen and thymus transport amino acids differently, and that the "T" lymphocytes from the spleen have membrane transport systems different from "T" lymphocytes from the thymus.     

Thymocyte and lymphocyte differentiation studied by means of acridine orange fluorescence microscopy

Summary Spleen, cervical lymph node and thymus of guinea pigs, half day, three weeks and nine months of age were investigated in fluorescent light after staining with acridine orange. Four varieties of lymphocytes have been found as differentiated presumably by the color of DNA of their nuclei. Two varieties are present in the thymus. The cortical thymocytes differ distinctly in color from those in the medulla. The splenic lymphocytes also display different nuclear color from those which are present in the cervical lymph nodes. The problems of the structure and function of the thymus have been discussed.     

Selective localization of tumor-immune spleen cells at the tumor challenge site after adoptive transfer of line 10 tumor immunity in strain 2 guinea pigs

In this study, the distribution of immune spleen cells was investigated after adoptive transfer of immunity in inbred strain 2 guinea pigs. Spleen cells obtained from line 10 immune donor animals became specifically restimulated in vitro with 3 M KCl-extracted line 10 soluble proteins, but not with 3 M KCl-extracted line 1 or liver proteins. After 4 days culture in vitro, these specifically restimulated immune spleen cells retained their antitumor activity in vivo after adoptive transfer. The specifically restimulated immune spleen cells were radiolabeled with [3H]thymidine, 1 X 10(8) viable cells were adoptively transferred in tumor-bearing guinea pigs, and their distribution was investigated. As controls for the specific localization of the immune cells at the line 10 tumor, the presence of labeled cells was studied in the contralateral transplanted line 1 hepatoma as well as in cellular inflammatory reactions elicited by injection with incomplete Freund's adjuvant (IFA) and complete Freund's adjuvant (CFA). A significantly higher localization of the labeled immune spleen cells in the line 10 tumor and the first and second draining lymph nodes of the line 10 challenge site were found when compared to the influx of these cells in the line 1 tumor and the nontumor antigen-related inflammatory reactions. Because our immune donor animals were immunized with a mixture of line 10 cells and BCG, these animals are immune to both. Line 10 immune spleen cells were restimulated in vitro with PPD and were radiolabeled. These PPD-restimulated immune spleen cells showed no preferential localization at the line 10 tumor challenge site but, as expected, a tendency for localization at the CFA (H37Ra) injection site. Furthermore, PPD-reactive spleen cells from BCG-immunized guinea pigs showed a significantly higher accumulation at the CFA injection site compared to the IFA injection site and the line 10 and line 1 tumor challenge site. From the results, it is concluded that line 10 tumor-immune and BCG-immune spleen cells are two distinct cell populations, and that the existence of cross-reacting antigens between BCG and the line 10 hepatocarcinoma are of no importance for the rejection of the line 10 tumor by immune spleen cells.