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PubMed1994

Role of GH and IGF-I in the regulation of IGF-I, IGF-I receptor and IGF binding protein gene expression in the rat spleen.

To characterize the expression of the IGF-I system in the spleen and its role in spleen growth, we have studied the effect of hypophysectomy and the action of either GH or IGF-I treatment on the expression of several components of the IGF system in the rat. Female Sprague-Dawley rats were hypophysectomized (Hx) on postnatal day 50, and five animals each received twice-daily sc injections of saline, bovine GH (bGH; 84 micrograms/animal/day), or recombinant human IGF-I (rhIGF-I; 125 micrograms/animal/day) for 11 days. Compared to sham-operated controls, Hx animals exhibited a reduction in both body (192.6 +/- 5.6 g (mean +/- S.E.M.) vs. 268.6 +/- 6.0 g; P < 0.001) and spleen weights (0.42 +/- 0.03 g vs. 0.84 +/- 0.06 g; P < 0.001). The reduction in body and spleen weights in Hx animals was partially prevented by both bGH and rhIGF-I. Body weights were 234.2 +/- 5.3 g (P < 0.001) after bGH and 213.8 +/- 6.3 g (P < 0.05) after rhIGF-I. Spleen weights were 0.56 +/- 0.048 after bGH P < 0.01 and 0.53 +/- 0.05 g after rhIGF-I (P < 0.05). Serum GH and IGF-I levels were markedly reduced in Hx animals and bGH partially maintained IGF-I levels. Hypophysectomy reduced spleen IGF-I mRNA levels (30.6 +/- 7.5% of control values; P < 0.05) and this reduction was prevented by bGH (96.6 +/- 24.2%; NS) but not by rhIGF-I (39.9 +/- 5.0% NS vs. Hx). There were no changes in GH receptor or IGF-I receptor mRNA levels in Hx or bGH or rhIGF-I-treated animals. When IGF-I binding protein (IGFBP) mRNA levels were studied under these conditions, we found that IGFBP-1 mRNA was not detected in spleen; IGFBP-2 mRNA levels were reduced in Hx rats (67.9 +/- 7.4% of control values, P < 0.05) and bGH treatment prevented this reduction (95.5 +/- 12.2%, NS). IGFBP-3 mRNA levels were not affected by hypophysectomy or by bGH treatment, but were reduced in rhIGF-treated rats (69.6 +/- 3.0%, P < 0.05). On the other hand, IGFBP-4 mRNA levels were increased in Hx rats (136.4 +/- 15.9% of control values, P < 0.05) and bGH treatment prevented this increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Spleen and portal lymphnode pathology in fatal drug addiction.

Spleen and portal lymphnode sections from 86 drug addicts submitted for medico-legal autopsy at the Institute of Forensic Medicine in Copenhagen in the year 1979 were studied together with tissue sections from 24 "normal" persons. In 70% of the drug addicts the spleen weight was more than 200 g, and in 71% portal lymphnode hyperplasia was found. Birefringent foreign material was found in spleen tissue of drug addicts in 72% and in portal lymphnode tissue in 44%. Signs of antigen stimulation in both spleen and portal lymphnode tissue evaluated by the number of germinal centre and plasma cells were found in more than 80% of the drug addicts compared with about 20% of the "normal" persons. The results were related to anamnestic information of duration of drug abuse, to the spleen weight, to the occurrence of birefringent material and to the liver changes. Examination of lysozyme and immunoglobulin containing cells using the indirect preoxidase technique was performed in a total of 72 cases of spleen tissue, 59 cases of portal lymphnode tissue from drug addicts, 24 cases of spleen tissue and 18 of portal lymphnode tissue from "normal" persons. Lysozyme, IgM and IgG containing cells were found significantly more often among drug addicts than "normal" persons. The results indicate that the splenomegalia and the portal lymphnode hyperplasia often found in drug addicts are caused by continuous antigen stimulation due to repeated injections of various antigens.

Adolescent↗
PubMed1992

Liver and spleen phagocytic depression after peripheral ischemia and reperfusion.

Liver and spleen phagocytic clearance of blood-borne microparticulate tissue debris and products of intravascular coagulation after trauma and surgical injury is an important mechanism to limit the deposition of debris in the pulmonary vascular bed. Plasma fibronectin (pFn) modulates this clearance process. We evaluated the effect of a localized peripheral ischemia and reperfusion injury on liver and spleen phagocytic function. Male rats (250 to 350 g) underwent 4 hours of tourniquet-induced bilateral hindlimb ischemia, followed by 18 hours of reperfusion after release of the tourniquet. Rats subjected to ether anesthesia alone or anesthesia followed by groin incision without ischemia were the control and sham groups, respectively. Reticuloendothelial (RE) phagocytic function was assessed at 15 minutes and 18 hours after the start of reperfusion by the in vivo liver and spleen removal of blood-borne iodine 125 (125I)-test microparticles, which were coated with gelatin (denatured collagen) to enhance their interaction with pFn. Liver and spleen particle uptake in control and sham rats was similar. In contrast, after 4 hours of ischemic injury with 15 minutes of reperfusion, we observed a 30% to 40% decrease (p less than 0.05) in liver and spleen particle uptake as compared with sham controls with partial restoration of this removal mechanism by 18 hours. This depression in liver and spleen phagocytic function was associated with a significant (p less than 0.05) increase in the deposition of the 125I-test particles in the lung. RE depression was not due to a deficiency of pFn; indeed, a marked elevation (588 +/- 12 micrograms/mL versus 1,083 +/- 40 micrograms/mL) of pFn was observed by immunoassay over the 18-hour reperfusion interval. Comparative bioassay of humoral (opsonic) versus cellular (Kupffer's cell) activity revealed that Kupffer's cells in livers from controls or ischemia-reperfusion rats exhibited normal phagocytic function when incubated in plasma harvested from either control or 4-hour ischemic rats. The opsonic activity of plasma harvested after ischemia and reperfusion was also more than adequate, consistent with the immunoassay analysis. Thus, the impaired liver and spleen clearance mechanism after peripheral ischemia and reperfusion injury did not appear to be due to either a macrophage cellular deficit or a lack of pFn. This clearance depression may be mediated by splanchnic malperfusion, which is known to develop after peripheral ischemia and reperfusion and associated soft tissue injury.

Animals↗
PubMed1997

Effects of varying the type of saturated fatty acid in the rat diet upon serum lipid levels and spleen lymphocyte functions.

To obtain further information about the effects of specific dietary saturated fatty acids, weanling male rats were fed for 6 weeks on low fat (7.7% by weight) or high fat (17.8% by weight) diets which differed according to the principal fatty acids present. The diets were rich in caprylic and capric acids (medium chain triacylglycerols; MCT), lauric acid, palmitic acid at the sn-1(3) position, palmitic acid at the sn-2 position or stearic acid. The total proportions of saturated (42-46%), monounsaturated (36%), n-6 polyunsaturated (15%) and n-3 polyunsaturated (2.2%) fatty acids were the same in all diets. Serum cholesterol concentrations were not different among rats fed the different diets, except that the concentration in the serum of rats fed the high fat diet with palmitic acid in the sn-2 position was high. This was reflected in higher HDL and LDL cholesterol concentrations in the serum of animals fed this diet. Triacylglycerol (TAG) concentrations tended to be higher in the serum of rats fed the low fat diets compared with those fed the high fat diets. They were lowest in the serum of MCT-fed rats irrespective of the level of fat in the diet and were highest in the serum of rats fed the low fat diet rich in stearic acid. These differences were due to lower chylomicron and VLDL TAG concentrations in the serum of MCT-fed rats and higher chylomicron and VLDL TAG concentrations in the serum of low fat, stearic acid-fed animals. The fatty acid compositions of the serum and of spleen lymphocytes were influenced by that of the diet fed. The ex vivo proliferation of lymphocytes from the spleens of rats fed the high fat diet rich in palmitic acid at the sn-2 position was greater than that of lymphocytes from animals fed the other diets. Natural killer (NK) cell activity tended to be lower for spleen lymphocytes from rats fed high fat diets than for those fed low fat diets irrespective of the principal saturated fatty acid present. NK cell activity was highest for spleen lymphocytes from animals fed the diets rich in palmitic acid and was lowest for those from animals fed the high fat diet rich in stearic acid. Spleen lymphocytes from the latter animals had the lowest proportion of CD16+ cells, a marker for NK cells. Thus, this study shows that the type of saturated fatty acid present in the diet not only has subtle effects upon blood lipid and lipoprotein levels but can significantly affect lymphocyte functions. Spleen lymphocyte NK cell activity is decreased as the fat content of the diet increases. NK cell number and activity are reduced by a high fat diet rich in stearic acid. Spleen lymphocyte proliferation is enhanced by palmitic acid-rich diets, particularly if palmitic acid is in the sn-2 position of dietary TAG.

Animals↗
PubMedDate not supplied

Differentiation of Th1 and Th2 cells in lymph nodes and spleens of mice during experimental autoimmune uveoretinitis.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease that can be elicited in susceptible rodent strains by immunization with a retinal autoantigen, such as interphotoreceptor retinoid-binding protein (IRBP). In this study, we investigated whether there is a correlation between inflammation in the eye and T-helper (Th)1- and Th2-type responses in the lymph nodes and the spleen after immunization of B10.A mice with IRBP. METHODS: B10.A mice were immunized with IRBP emulsified with complete Freund's adjuvant (CFA), and eyes were then enucleated for histological examination of EAU at 1, 2, 4, 6, or 8 weeks after immunization. In addition, lymph node cells and spleen cells were collected, and cultured with IRBP to measure T-cell proliferation responses and Th1-type (interleukin [IL]-2, interferon [IFN]-gamma), Th2-type (IL-4, IL-10) cytokine production. RESULTS: Pathologically, severe ocular inflammation occurred 2 weeks after IRBP immunization, persisted for 2 weeks, and then gradually resolved. Interleukin-2 and IFN-gamma production were observed in draining lymph node cells at 1 and 2 weeks after IRBP immunization. Those responses then diminished, whereas IFN-gamma production by spleen cells was observed from week 1, peaked at week 4, and gradually decreased. Alternatively, significant production of IL-4 or IL-10 by draining lymph node cells was not detected at any time point. Both IL-4 and IL-10 production by spleen cells was observed at week 6. CONCLUSIONS: Th1-type responses were observed early in draining lymph nodes, then in the spleen after IRBP immunization. The levels of IFN-gamma production by spleen cells reflected the severity of EAU, confirming their pathogenic role in this disease. Th2-type responses were generated in the spleen only as the disease receded, suggesting a role for Th2 cells in the spontaneous termination of EAU.

Animals↗
PubMed1998

Photofrin increases murine spleen cell transferrin receptor expression and responsiveness to recombinant myeloid and erythroid growth factors.

When given to normal male mice, the photochemotherapeutic agent Photofrin (porfimer sodium), a complex mixture of monomeric and oligomeric non-metallic porphyrins, significantly increased relative spleen weight, spleen cell numbers, DNA replication, levels of granulocyte-macrophage and erythroid progenitors and cellular responsiveness to different recombinant (r) myeloid growth factors. In contrast, monomeric hematoporphyrin had no effect on spleen weight, cellularity, erythroid progenitor levels or spleen cell cycle status. Photofrin significantly increased spleen cell expression of the receptor (CD71) for the iron transport protein transferrin by 72 h post-injection but did not affect levels of a receptor (CD25) for the T-cell growth factor interleukin-2 (IL-2) or spleen cell responsiveness to rIL-2. Further evidence of increased splenic erythropoietic activity in Photofrin-treated mice was provided by double color flow cytometric studies which indicated that the treatment elevated the number of nucleated spleen cells recognized by the erythroid lineage-specific monoclonal antibody TER-119. A majority of TER-119+ cells also expressed CD71 and the heat stable antigen, a marker of developing hematopoietic cells as well as mature erythrocytes. The capacity of Photofrin to stimulate murine hematopoietic activity appears to be a property not shared by other porphyrin photosensitizers.

Animals↗
PubMed1997

Transforming growth factor-beta isoforms in the developing chicken intestine and spleen: increase in transforming growth factor-beta 4 with coccidia infection.

Expression of transforming growth factor-betas 2, 3 and 4 (TGF-beta) in the developing chicken intestine and spleen was investigated using specific cDNA probes and antibodies for the different TGF-beta isoforms. Coordinate expression of the mRNAs for TGF-beta s 2, 3 and 4 was detected in the embryonic intestine by 8 days, with maximal expression of the mRNAs for TGF-beta s 2 and 4 occurring at 12 and 19 days, respectively, while expression of TGF-beta 3 mRNA remained constant during this time. While specific antibodies for TGF-beta s 2, 3 and 4 could detect only weak immunohistochemical staining of the intestinal epithelium in 4-, 12- and 16-day-old embryos, intense staining for TGF-beta s 2, 3 and 4 was detected in the tips of the intestinal villi of 19-day-old embryos. In the spleen, expression of the mRNAs for TGF-beta s 2 and 3 increased in the newly hatched chick compared with the embryo and then decreased in the adult to levels that were lower than in the embryo; expression of TGF-beta 4 mRNA increased progressively with developmental age, with expression in the adult spleen being significantly higher than in the embryonic and hatchling spleen. Immunohistochemical staining of spleens showed a selective increase in the level of reactive TGF-beta 4 with increasing developmental age, while staining for TGF-beta s 2 and 3 was constant during development. After infection of 1-month-old chickens with coccidian parasite, expression of TGF-beta 4 mRNAs increased 5-8-fold in intestinal intra-epithelial lymphocytes and 2.5-fold in spleen cells, while expression of the mRNAs for TGF-beta s 2 and 3 remained constant in these cells. The results of this study suggest that TGF-beta may play a role in development of the intestine and spleen in the chicken and that TGF-beta 4 in particular increases after infection of coccidia in the chicken.

Animals↗
PubMed2003

Comparison of CTL reactivity in the spleen and draining lymph nodes after immunization with peptides pulsed on dendritic cells or mixed with Freund's incomplete adjuvant.

OBJECTIVE: To compare CTL reactivity in the spleen and the draining lymph nodes (LN) from C57BL/6 mice after immunization with self and non-self peptides pulsed on autologous dendritic cells (DC) or mixed with Freund's incomplete adjuvant (FIA). METHODS: Peptides showing high to low binding affinities for H-2 Kb/Db were emulsified in FIA or pulsed on bone marrow (BM)-derived DC and injected subcutaneously into C57BL/6 mice. Eight days later, the mice were sacrificed and cell suspensions were prepared from the spleen and draining LN. Splenocytes or LN cells were cultured for 5 days with irradiated syngeneic spleen cells (as APCs) pulsed with the appropriate peptide in vitro. 51Cr-release assay using peptide pulsed target cells was used to detect CTL reactivity. RESULTS: Both self and non-self peptides can induce specific CTL responses with the adjuvant FIA and DC. Peptide pulsed DC were found to be more effective than peptides mixed with FIA to induce specific CTL responses towards non-self peptides and can induce much stronger responses in the spleen than in the draining LN both for non-self and self peptides. Self peptides emulsified in FIA generated the strongest responses in the draining LN, whereas non-self peptides mixed with FIA generated the strongest response in the spleen. CONCLUSIONS: DC-based immunization with non-self and self peptides is more efficient than immunization based on peptides mixed with FIA. DC-based immunization focuses the CTL response towards the spleen. Immunization based on FIA focuses the response against self peptides towards the draining LN and non-self peptides towards the spleen.

Amino Acid Sequence↗
PubMed1990

Staphylococcal enterotoxin A induced interferon (IFN)-gamma production in spleen cells from BCG-immunized mice: the IFN production is dependent on leukotriene C4 but not dependent on interleukin 2.

In our previous paper, we showed that IFN was induced in sera by injection of staphylococcal enterotoxin A (SEA) in Bacillus Calmette-Guérin (BCG) immunized C57BL/6 (B6) mice. In analyzing the phenomenon in vitro, we showed that SEA induced IFN-gamma in the supernatant of the spleen cell culture from BCG immunized B6 mice and that leukotriene C4 (LTC4) from BCG activated macrophages in the spleen was involved in the IFN production from Ly 1+ T cells. On the other hand, interleukin-2 (IL-2) has reported to play an important role in the regulation of synthesis of IFN-gamma by T cells. In the present study, we examined whether IL-2 is involved in SEA-induced IFN production. The result showed that the SEA-induced IFN-gamma production was observed in spite of suppression of SEA-induced IL-2 production in spleen cells from BCG-immunized B6 mice. On the contrary, the depressed IFN production was observed in spite of high SEA-induced IL-2 production in spleen cells from their control mice. On the other hand, LTC4 production was 8 times higher in spleen cells from BCG-immunized B6 mice, high producer of SEA-induced IFN, than in that from BCG-immunized C3H mice, the low producer. We also observed that the IFN and the LTC4 production of spleen cells from BCG-immunized B6 mice was suppressed in the presence of caffeic acid and nordihydroguaiaretic acid, non-specific lipoxygenase inhibitors, and that LTC4 augmented the IFN production of normal B6 mouse spleen cells in the presence of 2-mercaptoethanol. Therefore, involvement of LTC4 rather than of IL-2 was supported in our experimental system.

Animals↗
PubMed1987

Drug-induced tolerance to allografts in mice. X. Augmentation of split tolerance in murine combinations disparate at both H-2 and non-H-2 antigens by the use of spleen cells from donors preimmunized with recipient antigens.

In a fully allogeneic murine combination of C3H/HeSlc (C3H) (H-2k) and C57BL/6CrSlc (B6) (H-2b), C3H mice were primed i.v. with 1 X 10(8) spleen cells from B6 mice preimmunized i.v. with 5 X 10(7) C3H spleen cells and then were given i.p. 200 mg/kg cyclophosphamide (CP) 2 days later (Im-B6-Sc plus CP group). The tolerant state in those recipient mice was compared with that in mice made tolerant conventionally with 1 X 10(8) naive B6 spleen cells plus 200 mg/kg CP (naive-B6-Sc plus CP group). B6 skin was rejected in an almost normal fashion in both the naive-B6-Sc plus CP group and the Im-B6-Sc plus CP group. However, EL4 tumor allografts (B6 origin) inoculated after complete rejection of B6 skin grafts were specifically accepted in both groups. Moreover, the tumor growth in the Im-B6-Sc plus CP group was faster than that in the naive-B6-Sc plus CP group. Mixed lymphocyte reaction, cytotoxic T lymphocyte activity, and antibody production against the tolerogen were depressed more profoundly in the Im-B6-Sc plus CP group than in the naive-B6-Sc plus CP group. These observations were consistent with the results from tumor allografting. The other immunological parameters examined in the present study, including helper T cell activity and delayed foot-pad reaction, were retained in the Im-B6-Sc plus CP group at the same levels as in the naive-B6-Sc plus CP group. These observations were consistent with the results from skin allografting. In conclusion, tumor allograft tolerance was made more profound by the use of spleen cells from donors preimmunized with recipient antigens as the tolerogen than by the use of naive spleen cells. However, skin allograft tolerance was not achieved at all by these same treatments. The contribution of graft-versus-host disease to this phenomenon was excluded by the chimeric analysis in AKR/JSea (H-2k) mice given the preimmunized (with AKR antigens) B6 spleen cells plus CP. These results strongly support the existence of a less proliferative lymphocyte population which does not evoke cell divisions to mature even after the strong stimulation with the preimmunized spleen cells and is resistant to tolerance induction.

Animals↗
PubMed1989

The effect of T cell depletion from spleen cell allografts on graft-versus-host disease and long-term immune reconstitution in H-2 haplotype-identical murine combinations.

Graft-versus-host disease (GVHD) and states of immune reconstitution in allogeneic chimera mice across minor histocompatibility antigens were analyzed in excess of 9 months after injecting AKR/JSea (AKR) spleen cells into irradiated C3H/HeSlc (C3H) mice. When T cell-depleted AKR spleen cells were used as inoculum cells, neither graft failure nor GVHD was seen for 9 months postgrafting in the C3H mice irradiated with 660 rad or more. In an AKR - C3H (850 rad) model, Thy1.1+ or L3T4+ T cell depletion from donor AKR spleen cells abolished both acute and chronic GVHD in lethally irradiated C3H mice. Lyt2+ T cell depletion, however, resulted in acute and chronic GVHD in more than half of the recipient C3H mice. Moreover, actual existence of donor (AKR)-type T cells with L3T4 phenotype, but not Lyt2 phenotype, was always observed in the spleen of the C3H mice suffering from acute GVHD. In addition, the C3H mice that were irradiated with 850 rad, grafted with AKR spleen cells depleted of Lyt2.1+ T cells, escaped from acute GVHD and survived for more than 10 mo postgrafting, showed impaired activities of immune responses such as delayed footpad reaction to sheep red blood cells, antibody production tested by IgM plaque forming cells and reactivity to an intracellular bacterium. Listeria monocytogenes as compared with the C3H mice reconstituted with syngeneic C3H spleen cells or Thy1.1+ or L3T4+ T cell-depleted AKR spleen cells. These results suggest that L3T4+ T cells, rather than Lyt2+ T cells, contained in the grafted cells not only cause acute GVHD but also a long-term immunodeficient state (chronic GVHD) in recipient mice in the H-2-identical murine combinations examined here.

Animals↗
PubMed1991

The spectrum of liver and spleen injuries in children: failure of the pediatric trauma score and clinical signs to predict isolated injuries.

STUDY OBJECTIVES: To determine whether potentially life-threatening intra-abdominal injuries occur in the absence of multisystem trauma in children, and to determine the usefulness of physical examination and a pediatric triage score in the assessment of liver and spleen injuries in children. DESIGN: Retrospective study. SETTING: Admissions to a children's hospital from October 1982 through September 1989 who were found to have liver or spleen injuries. Seventy-seven patients were identified; 55 were male, 22 were female. Mean age was 9 years and 3 months (+/- 56 months) with a range of 22 months to 20 years, 4 months. Clinical signs were recorded and a Pediatric Trauma Score (PTS) and Injury Severity Score (ISS) were calculated for each patient. MEASUREMENTS AND MAIN RESULTS: Fifty-four patients (70%) had a spleen injury, 18 (23%) had a liver injury, and five (7%) had both liver and spleen injuries. Patients were managed without surgery in 63 of 77 cases (82%); two died. Fifty-one of 77 patients (66%) received an ISS of 18 or less; 26 patients (34%) received a score of more than 18. Forty-four of the 51 patients (86%) with an ISS of 18 or less had a normal pulse (120 or less); 48 of the 51 (94%) had a normal systolic blood pressure (90 mm Hg or more). A strong negative correlation (r = -0.80; P = .001) was found between the two scores for children with multiple severe injuries (ISS of more than 18); there was poor correlation (r = -0.04; P greater than .05) between the two scores for isolated liver or spleen injury (ISS of 18 or less). CONCLUSIONS: We conclude that liver or spleen damage may be present in children without other injuries and must be considered with a high index of suspicion, and that neither the initial clinical findings nor the PTS reliably predicts liver or spleen injuries in children with focal abdominal injuries.

Adolescent↗
PubMed2001

Bilateral dissected spleens and thymuses in rodents exhibit homogeneity in leukocyte markers.

Histopathologic evaluation and/or archiving of sections of spleen or thymus from all study animals may be mandated by study protocol (e.g., Toxic Substances Control Act-compliant studies). In such cases, whole spleen or thymus is not available for immunophenotyping. It has not been previously demonstrated that immunologic data representative of whole organs can be reliably obtained using a section of the spleen or using one thymic lobe. Light-scatter characteristics and immune cell-surface antigen expression were therefore compared in the right and left halves of the spleen and in the right and left thymic lobes of young adult female C57B1/6 mice and Sprague-Dawley rats. Antigens compared were: mouse spleen - CD11b, CD45R, CD90; rat spleen - CD11b, CD45RA, Pan-T/Ox-52; mouse and rat thymus - CD4, CD8a. There were no significant differences in distribution of cells by size or by expression level for any of these antigens when the right part of the organs was compared to the left part. These data indicate that use of entire spleen or both thymic lobes is not required to reliably quantify resident immune cell subpopulations.

Animals↗
PubMed2002

Blood and spleen lymphocytes as targets for immunotoxic effects in the rat--a comparison.

Traditionally, immunotoxicological studies in the rat have been performed by measuring the effect of chemical substances on spleen lymphocytes in vivo and in vitro. However, rat blood lymphocytes may be more relevant than spleen cells for comparison with human blood lymphocytes. Further, lymphocytes in blood may be a more sensitive indicator of immunotoxic effects than spleen lymphocytes. Finally, in longitudinal studies peripheral blood specimens can be collected repeatedly from the same animals, thereby reducing the number of animals sacrificed and, possibly, experimental variation. We compared blood and spleen lymphocyte parameters in rats treated with a single dose of the immunosuppressant cyclophosphamide (CY), monitoring effects on blood and spleen lymphocytes by immunophenotyping. We also performed repeated bleedings to demonstrate the feasibility of following the time course of induced changes in the same animals. Immunophenotyping as well as total mononuclear cell counts consistently showed as large or lager effects of CY in blood lymphocytes than in spleen cells. Further, the measured effects in blood lymphocytes became statistically significant at an earlier time point, compared to spleen cells. Repeated bleedings of the same animals illustrated that blood specimens drawn from a peripheral vein give sufficient numbers of cells to perform immunotoxicological tests.

Animals↗
PubMed2002

Roles of spleen and liver in development of the murine hematopoietic system.

OBJECTIVE: Hematopoietic stem cells (HSCs) and colony-forming progenitor cells (CFCs) are believed to migrate from liver to bone marrow (BM) around the time of birth, where they remain throughout the animal's life. Although in mice the spleen is also a hematopoietic organ, neither the origin nor the contribution of spleen HSCs to hematopoietic homeostasis has been assessed relative to that of BM HSCs. To investigate these issues we quantitated CFC and HSC activity in the spleen, BM, peripheral blood, and liver of the mouse during ontogeny. METHODS: CFCs were assessed by clonogenic colony formation, and HSCs by long-term reconstituting ability. RESULTS: CFCs gradually increased in the BM and decreased in the liver with age. Increased prevalence of CFCs in fetal and pup blood occurred at day (d) 12 postcoitus (pc) and during the period of d16 pc to 4d postbirth, corresponding to the times when hematopoietic cells migrate from the yolk sac and/or aorta-gonad-mesonephros (AGM) to the fetal liver and from the neonatal liver to the BM, respectively. In the spleen, CFCs displayed two peaks of activity at 2d and 14d-15d postbirth. Spleen HSCs also fluctuated during this time period. Neonatal splenectomy did not alter CFC or HSC frequencies in the BM, but CFCs increased in the livers of splenectomized mice. CONCLUSIONS: These data demonstrate that the liver may act as a site of extramedullary hematopoiesis in the neonate, especially in the absence of the spleen, and imply that the spleen, BM, and liver cooperatively contribute to hematopoietic homeostasis.

Animals↗
PubMed2001

Identification of tyrosine hydroxylase gene expression in rat spleen.

This study was aimed to identify tyrosine hydroxylase (TH) gene expression in the rat spleen under basal and stress conditions. Using the reverse transcription polymerase chain reaction we did not detect TH mRNA in rat spleen either in control, or immobilized animals. Semi-nested PCR revealed a clear signal, demonstrating that TH mRNA is formed in the spleen, although in low abundance. We also detected both, TH immunoreactive protein and TH activity in the rat spleen that were in higher abundance than expected from the mRNA levels. This study identifies, for the first time, TH gene expression in rat spleen. Since TH protein and activity are present in the spleen in much higher abundance compared to corresponding mRNA, the majority of TH protein is most probably supplied by the sympathetic innervation of spleen.

Animals↗
PubMed2002

Stress increases gene expression of phenylethanolamine N-methyltransferase in spleen of rats via pituitary-adrenocortical mechanism.

Gene expression of phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing conversion of norepinephrine to epinephrine, has been detected in rat spleen using the reverse transcription polymerase chain reaction. PNMT identity was subsequently verified by Southern blots. Localization of the spleen cells responsible for the PNMT gene expression was investigated by the in situ hybridization and PNMT mRNA was found to be present in the white pulp. The hypothesis that stress may produce an increase in PNMT gene expression in rat spleen was tested and a robust rise in the relative abundance of PNMT mRNA levels was observed after a single or repeated immobilization (about 80%). Adrenalectomy or hypophysectomy completely prevented the immobilization-induced increase in spleen PNMT mRNA levels, suggesting that stress-induced PNMT gene expression in the spleen is regulated predominantly via pituitary-adrenocortical axis. In control animals, however, spleen PNMT was not significantly affected by the ectomies and therefore basal PNMT gene expression might be regulated by different mechanism(s).Thus, PNMT gene expression in the rat spleen is exaggerated by stress stimuli, suggesting its role in physiological regulations.

Adrenal Cortex↗
PubMed1998

Developmental changes of cytochrome P450 dependent monooxygenase functions after transplantation of fetal liver tissue suspension into spleens of adult syngenic rats.

Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher 344 inbred rats. Animals were sacrificed at 3 days, 1, 2, 4 weeks, and 2, 4 and 6 months after transplantation and cytochrome P450 (P450) dependent monooxygenase functions in spleen and liver 9000 g supernatants were assessed by measuring three model reactions for different P450 subtypes: ethoxyresorufin O-deethylation (EROD; mainly 1A), ethoxycoumarin O-deethylation (ECOD; predominantly 1A, 2A, 2B) and ethylmorphine N-demethylation (END; mainly 3A). Values of transplant recipients were compared to those of sham operated and age matched control rats. Spleen weights were significantly higher in transplanted rats, compared to controls or sham operated animals, but there was no influence of the transplants within the spleens on liver weights. With fetal livers at the 21st day of gestation, the day of transplantation, a weak EROD and ECOD, but no END activity was seen. Spleens of controls or sham operated animals displayed nearly no P450 mediated monooxygenase functions. In the explant containing spleens a significant and increasing EROD activity was found from 4 weeks after surgery on and an ECOD activity already 2 weeks after transplantation. END was only slightly enhanced at 6 months after surgery. The livers of all three groups of rats displayed normal EROD, ECOD and END activities. Transplantation of fetal liver tissue suspensions into the spleens did not influence the P450 dependent monooxygenase functions within the livers of the animals. From these results it can be concluded that intrasplenically transplanted liver cells originating from syngenic fetal liver tissue suspensions proliferate and differentiate within the host organs. They display P450 dependent monooxygenase functions with some developmental changes during the observed time period of 6 months.

Animals↗