Interaction of aging with lipoxygenase deficiency initiates hypersplenism, cardiac dysfunction, and profound leukocyte directed non-resolving inflammation.

In the process of physiological cardiac repair, splenic leukocyte-activated lipoxygenases (LOXs) are essential for the biosynthesis of specialized pro-resolving lipid mediators as a segment of an active process of acute inflammation in splenocardiac manner. In contrast, young 12/15LOX-/- mice use a compensatory mechanism that amplifies epoxyeicosatrienoic acid mediators after myocardial infarction, improving cardiac repair, function, and survival. Next, we tested whether deletion of 12/15LOX impacted the genesis of chronic inflammation in progressive aging. To test the risk factor of aging, we used the inter-organ hypothesis and assessed heart and spleen leukocyte population along with the number of inflammation markers in age-related 12/15LOX-/- aging mice (2 months, 6 months, 13 months) and compared with C57BL/6 J (WT; wild type) as controls (2 months). The 12/15LOX-/- aging mice showed an age-related increase in spleen mass (hypersplenism) and decreased marginal zone area. Results suggest increased interstitial fibrosis in the heart marked with the inflammatory mediator (PGD2) level in 12/15LOX-/- aging mice than WT controls. From a cellular perspective, the quantitative measurement of immune cells indicates that heart and spleen leukocytes (CD11b+ and F4/80+ population) were reduced in 12/15LOX-/- aging mice than WT controls. At the molecular level, analyses of cytokines in the heart and spleen suggest amplified IFN-γ, with reduced COX-1, COX-2, and ALOX5 expression in the absence of 12/15LOX-derived mediators in the spleen. Thus, aging of 12/15LOX-/- mice increased spleen mass and altered spleen and heart structure with activation of multiple molecular and cellular pathways contributing to age-related integrative and inter-organ inflammation.

Impact of total splenectomy on peripheral lymphocytes and their subsets in patients with hypersplenism associated with cirrhotic portal hypertension.

To study the impact of total splenectomy (TS) on peripheral lymphocytes and their subsets in patients with hypersplenism associated with cirrhotic portal hypertension (CPH). We studied 102 consecutive patients who received TS from January 2008 to January 2020 due to CPH-related hypersplenism. A similar number of healthy individuals are used as healthy controls (HC). The total lymphocyte counts and their percentages of B lymphocytes, total T lymphocytes (cluster of differentiation (CD)3+) and their subsets (CD4+, CD8+), and natural killer (NK) cells in preoperative peripheral blood samples in hypersplenism patients were significantly lower than that of the HCs (both P < 0.05). The total lymphocyte counts and percentages of B lymphocytes in peripheral blood were significantly increased 1 week and 1 month after TS when compared with the pre-TS values (P < 0.05). There was no significant difference in the percentages of NK cells before or after surgery (P > 0.05). However, the percentages of CD3+ cells was significantly higher 1 month after than before surgery (P < 0.001). The percentages of CD4+, and CD8+ T lymphocytes were significantly lower 1 week after surgery (P < 0.05), but they were significantly higher 1 month after surgery (P < 0.01). The CD4+:CD8+ ratio was not significantly different from those before surgery, and 1 week or 1 month after surgery (P > 0.05). Patients with hypersplenism associated with CPH were significantly immunosuppressed preoperatively. After TS, the total lymphocyte count and percentages of B lymphocytes, and total T lymphocytes and their subsets increased significantly, resulting in improved immune functions.

Abnormal glucose metabolism in patients with Fontan circulation: Unique characteristics and associations with Fontan pathophysiology.

BACKGROUND: Fontan patients exhibit a high prevalence of abnormal glucose metabolism (AGM). We aimed to characterize AGM and clarify its association with Fontan pathophysiology. METHODS: We prospectively evaluated AGM with plasma glucose dynamics [mg/dL; fasting glucose (FPG), and maximum glucose increase (PG-spike)] during oral glucose tolerance test and hemoglobin A1c (HbA1c) in 276 consecutive Fontan patients (aged 19 ±â€¯7 years). Of these, 176 patients had serial AGM assessments with a mean interval of 6.5 years. RESULTS: Initial analysis revealed a high prevalence of impaired glucose tolerance (38.4%) and diabetes mellitus (DM) (4.7%), and positive family history, high HbA1c, and high central venous pressure independently predicted presence of DM. HbA1c was independently determined by hypersplenism and presence of DM (P < .05). Serial assessments revealed an increased PG-spike and a decreased HbA1c (P < .001 for both). Prevalence of DM increased (6.3% to 10.3%), and positive family history, high liver enzymes, and AGM predicted new onset of DM (P < .05 for all). Twenty-one patients died during 7.1-year follow-up. FPG (P < .01) and PG-spike (P < .05) independently predicted all-cause mortality. Particularly, patients with FPG ≤ 74 and/or PG-spike ≥85 had a mortality rate 8.7 times higher than those without (P = .0129). CONCLUSIONS: AGM progressed even in young adult Fontan patients, and HbA1c showed limited predictive value for progression. Oral glucose tolerance test plays important roles in uncovering unique Fontan AGM as well as predicting all-cause mortality.

Spleen Regulates Hematopoietic Stem/Progenitor Cell Functions Through Regulation of EGF in Cirrhotic Hypersplenism.

BACKGROUND: Hematopoietic abnormality is a common cause of cirrhotic hypersplenism (CH) complications and death; it causes serious adverse effects and is associated with bleeding, anemia, infection in CH patients. However, the underlying mechanism is unclear. AIMS: We aimed to investigate the effects of the spleen on hematopoiesis and hematopoietic stem/progenitor cells (HSPCs) in CH patients. METHODS: Eleven CH patients were enrolled to assess the effects of the spleen on HSPC functions. Hematopoietic changes were examined by flow cytometry analysis. HSPC functions were detected with colony-forming assays and in vitro cell cultures. Enzyme-linked immunosorbent assay (ELISA) was used to test the concentration of epithelial growth factor (EGF). RESULTS: The number of HSPCs was decreased in CH patients and was rescued after splenectomy. Serum from CH patients dysregulated HSPCs function, and serum from splenectomy patients restored the dysregulated HSPC function in vitro. The concentration of EGF was decreased in CH patients and was restored to normal level after splenectomy. EGF rescued the dysregulated HSPCs function in vitro. CONCLUSIONS: The spleen can regulate the functions of HSPCs in CH patients by regulating EGF signaling. EGF may be a therapeutic target for CH treatment.

Splenectomy attenuates murine liver fibrosis with hypersplenism stimulating hepatic accumulation of Ly-6C(lo) macrophages.

BACKGROUND & AIMS: Splenectomy in cirrhotic patients has been reported to improve liver function; however the underlying mechanism remains obscure. In the present study, we investigated the mechanism using a murine model, which represents well the compensated liver cirrhosis. METHODS: C57BL/6 male mice were allowed to drink water including thioacetamide (TAA: 300 mg/L) ad libitum for 32 weeks. After splenectomy at 32 weeks, mice were sacrificed on days one, seven, and 28, respectively, while TAA-administration was continued. Perioperative changes in peripheral blood and liver tissues were analyzed. RESULTS: TAA treatment of mice for 32 weeks reproducibly achieved advanced liver fibrosis with splenomegaly, thrombocytopenia, and leukocytopenia. After splenectomy, liver fibrosis was attenuated, and macrophages/monocytes were significantly increased in peripheral blood, as well as in the liver. Progenitor-like cells expressing CK-19, EpCAM, or CD-133 appeared in the liver after TAA treatment, and gradually disappeared after splenectomy. Macrophages/monocytes accumulated in the liver, most of which were negative for Ly-6C, were adjacent to the hepatic progenitor-like cells, and quantitative RT-PCR indicated increased canonical Wnt and decreased Notch signals. As a result, a significant amount of ß-catenin accumulated in the progenitor-like cells. Moreover, relatively small Ki67-positive hepatic cells were significantly increased. Protein expression of MMP-9, to which Ly-6G-positive neutrophils contributed, was also increased in the liver after splenectomy. CONCLUSIONS: The hepatic accumulation of macrophages/monocytes, most of which are Ly-6C(lo), the reduction of fibrosis, and the gradual disappearance of hepatic progenitor-like cells possibly play significant roles in the tissue remodeling process in cirrhotic livers after splenectomy.

NF-κB p65 and c-Rel subunits promote phagocytosis and cytokine secretion by splenic macrophages in cirrhotic patients with hypersplenism.

Transcription factors of the nuclear factor-kappa B (NF-κB) family play a key role in various biological processes. In this study, we explored the role of NF-κB in the dysfunction of splenic macrophages in hypersplenism due to liver cirrhosis. By using confocal microscopic analysis, Western Blot, TransAM NF-κB ELISA, and chromatin immunoprecipitation (ChIP), we observed that NF-κB p65, p52, and c-Rel were activated in macrophages in patients with hypersplenism (hypersplenic macrophages). Transfection of hypersplenic macrophages with a κB/luciferase reporter plasmid showed that NF-κB complexes were functional. Using co-immunoprecipitation studies, we demonstrated that p65/c-Rel dimers were activated in hypersplenic macrophages. NF-κB activation inhibitor JSH-23 and the small interfering RNA (siRNA)-mediated p65, and c-Rel gene silencing significantly blocked phagocytosis and secretion in hypersplenic macrophages. Using promoter analysis and RNA interference, we found that many phagocytotic and hepatic fibrogenetic regulators, including interleukin (IL)-1α, IL-1ß, interferon-γ (IFN-γ), transforming growth factor-ß1 (TGF-ß1), and tumor necrosis factor-α (TNF-α), were regulated by NF-κB p65 and c-Rel in hypersplenic macrophages. Our findings demonstrate that NF-κB p65 and c-Rel play an important role in phagocytosis and secretion in hypersplenic macrophages. Activation of NF-κB p65 and c-Rel may be considered an important regulator of hypersplenism and liver cirrhosis.

Bone marrow biopsy findings in brucellosis patients with hematologic abnormalities.

BACKGROUND: Brucellosis can mimic various multisytem diseases, showing wide clinical polymorphism that frequently leads to misdiagnosis and treatment delay, further increasing the complication rates. In this study, we aimed to examine bone marrow biopsy findings in brucellosis cases presenting with hematologic abnormalities. METHODS: Forty-eight brucellosis cases were prospectively investigated. Complaints and physical examination findings of patients were recorded. Patients' complete blood count, routine biochemical tests, erythrocyte sedimentation rate, C-reactive protein and serological screenings were performed. Bone marrow biopsy and aspiration was performed in patients with cytopenia, for bone marrow examination and brucella culture, in accordance with the standard procedures from spina iliaca posterior superior region of pelvic bone. RESULTS: Of the 48 patients, 35 (73%) were female and 13 (27%) were male. Mean age was (34.8 ± 15.4) years (age range: 15 - 70 years). Anemia, leukopenia, thrombocytopenia and pancytopenia were found in 39 (81%), 28 (58%), 22 (46%) and 10 patients (21%), respectively. In the examination of bone marrow, hypercellularity was found in 35 (73%) patients. Increased megacariocytic, erythroid and granulocytic series were found in 28 (58%), 15 (31%) and 5 (10%) patients, respectively. In addition, hemophagocytosis was observed in 15 (31%) patients, granuloma observed in 12 (25%) and increased eosinophil and plasma cells observed in 9 (19%) patients. CONCLUSION: According to the results of our series, hemophagocytosis, microgranuloma formation and hypersplenism may be responsible for hematologic complications of brucellosis.

Expression of NALP3 in the spleen of mice with portal hypertension.

This study examined the mRNA expression of NALP3 in the spleen of the mice with hypersplenism due to portal hypertension (PH). The mouse hypersplenism models were established by oral administration of tetrachloromethane (2 mL/kg/week for 12 weeks by oral gavage). All the mice were randomly divided into a control group and an experimental group. The blood routine test was conducted, spleen index was calculated and spleen was histologically examined. Portal vein sera were taken for detection of the level of uric acid. The mRNA expressions of NALP3 and IL-1beta in the spleen were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the platelet count was significantly lower in the experimental group [(674 + or - 102) x 10(9)/L] than in the control group [(1307 + or - 181) x 10(9)/L] (P<0.05), while the spleen index was significantly higher [(9.83 + or - 1.36) microg/g] in the experimental group than in the control group [(4.11 + or - 0.47) microg/g] (P<0.05). The histopathological changes of spleen followed the pattern of congestive splenomegaly. No significant difference was found in the uric acid level in the portal vein between the control group and the experiment group. The mRNA expressions of NALP3 and IL-1beta were up-regulated significantly in the spleen in the experimental group as compared with those in the control group (P<0.05). It was concluded that NALP3 and IL-1beta may play important roles in the pathogenesis of hypersplenism.

MicroRNAome of splenic macrophages in hypersplenism due to portal hypertension in hepatitis B virus-related cirrhosis.

MicroRNAs (miRNAs) are a recently discovered class of post-transcriptional regulators of gene expression with critical functions in health and disease. Their role in the pathogenesis of hypersplenism, however, is completely unknown. To determine whether miRNA expression is altered in splenic macrophages associated with hypersplenism due to portal hypertension in hepatitis-B-virus (HBV)-related cirrhosis, we analyzed the entire miRNAome in macrophages from normal and portal hypertensive spleen samples by microarray and Real-Time PCR. In this study, we identified 99 miRNA differences in expression in splenic macrophages associated with hypersplenism due to portal hypertension in HBV-related cirrhosis. Among the miRNAs identified in this study, hsa-miR-615-3p was significantly up-regulated in hypersplenism. Dynamic changes in miRNA expression occurred during the pathogenesis of portal hypertension-induced hypersplenism in HBV-related cirrhosis. The miRNAs then are novel regulatory RNAs in hypersplenism in patients with HBV-related cirrhosis.

Sites and kinetics of donor thrombopoiesis following transplantation of whole bone marrow and progenitor subsets.

INTRODUCTION: Little is known about the sites and kinetics of thrombopoiesis following bone marrow transplant. The spleen is a site of hematopoiesis in a healthy mouse, and hematopoietic activity increases in response to stress. We hypothesized that the spleen is a major site of early post-transplant thrombopoiesis. METHODS: We transplanted whole bone marrow (WBM) or lineage depleted progenitor subsets fractionated based on expression of c-kit and Sca-1 from transgenic mice expressing green fluorescent protein into lethally irradiated C57BL/6 recipients. We also transplanted whole bone marrow cells into healthy and splenectomized mice. Post-transplant megakaryopoiesis was assessed by measuring circulating platelet number, percent donor-derived platelets, bone marrow cellularity, splenic weight, megakaryocyte size, and megakaryocyte concentration from hour 3 to day 28 post transplant. RESULTS: Following transplant, circulating donor-derived platelets were derived only from c-kit expressing subsets. Donor-derived platelets first appeared on post-transplant day five. Splenectomy reduced the number of these earliest circulating platelets. Splenic megakaryopoiesis increased dramatically from day 7-14 post-transplant. However, splenectomy accelerated platelet engraftment during this time frame. CONCLUSION: Overall, these results demonstrate that the first platelets are produced by c-kit expressing megakaryocyte progenitors in the bone marrow and spleen. After post-transplant day 5, the net effect of the spleen on thrombopoiesis is to slow engraftment due to immune effects or hypersplenism.

[Expression and significance of Toll-like receptor 4 of splenic macrophage in patients with hypersplenism due to portal hypertension].

OBJECTIVE: To observe the change in expression of Toll-like receptor 4 (TLR4) of splenic macrophage in patients with hypersplenism due to portal hypertension (PH), and investigate the role of TLR4 of splenic macrophage in hypersplenism. METHODS: Splenectomy was performed on 20 patients with hypersplenism due to PH (hypersplenism group) and 6 patients with rupture of spleen by trauma (control group) and the specimens of spleen were collected. The splenic macrophages were isolated by adhibit wall method. The expression of TLR4 of splenic macrophage was detected by immunohistochemical method (SABC), and the result was analyzed by the image analysis system. The phagocytosis of splenic macrophage was measured by chicken red blood cell (CRBC) phagocytosis assay. The level of serum endotoxin was detected before the operation by limulus assay. The results of these 2 groups were compared, and correlation analysis was made among different results of the hypersplenism group. RESULTS: The expression of TLR4 of splenic macrophage was 109 +/- 32 in the hypersplenism group, significantly higher than that of the control group (62 +/- 5, P < 0.01). The rate of phagocytosis and index of phagocytosis of splenic macrophage in the hypersplenism group were 12.6% +/- 3.0% and 0.146 +/- 0.035 respectively, both significantly higher than those of the control group (6.9% +/- 0.5% and 0.076 +/- 0.008 respectively, both P < 0.01) The level of endotoxin of the hypersplenism group was 0.28 EU/ml +/- 0.21 EU/ml, significantly higher than that of the control group (0.054 EU/ml +/- 0.014 EU/ml, P < 0.05). The rate of phagocytosis and the index of phagocytosis of splenic macrophage were notably positively correlated with the expression of TLR4 (r = 0.601, P < 0.01 and r = 0.553, P < 0.05), and the level of endotoxin (r = 0.724 P < 0.01 and r = 0.506, P < 0.05). The expression of TLR4 of splenic macrophage was positively correlated with the level of endotoxin (r = 0.525, P < 0.05). CONCLUSION: The expression of TLR4 of splenic macrophage in patients with hypersplenism due to PH was increased significantly. It may be one of the important factors of hypersplenism due to PH that "endotoxemia-->increase of expression of TLR4 of splenic macrophage (activation of TLR of splenic macrophage)-->increased destruction of red blood cells by macrophage".

Diffuse splenic Tc-99m MDP uptake in hypersplenic patient.

A 52-year-old woman with nonspecific left leg pain was examined by Tc-99m methylene diphosphonate (MDP) bone scintigraphy. The patient had been a marble quarry worker for 10 years and had developed chronic congestive heart failure secondary to pneumoconiosis. Her hemoglobin analysis and hematologic findings were interpreted as being consistent with sickle cell beta+ thalassemia and also hypersplenism. Bone scintigraphy showed intense and diffuse MDP accumulation in the enlarged spleen without ultrasonographic or radiologic evidence of calcification.

Role of elevated platelet-associated immunoglobulin G and hypersplenism in thrombocytopenia of chronic liver diseases.

BACKGROUND AND AIM: Thrombocytopenia typically worsens with the progression of liver disease and can become a major clinical complication. Several mechanisms that contribute to thrombocytopenia have been proposed, including hypersplenism accompanied by increased platelet sequestration, platelet destruction mediated by platelet-associated immunoglobulins (PAIgG), and diminished platelet production stimulated by thrombopoietin (TPO). The purpose of the present study was to evaluate the role of each of these mechanisms in patients with liver disease-associated thrombocytopenia. METHODS: Twenty-nine patients with liver cirrhosis (LC), 20 of whom were hepatitis C virus (HCV)-seropositive, 29 chronic hepatitis (CH) patients, 24 of whom were HCV-seropositive, and 16 control patients without liver or hematopoetic disease were enrolled in this study. Serum TPO levels, PAIgG, and liver-spleen volumes were determined and correlation analyses were performed. RESULTS: No differences in serum TPO levels were observed among the three groups. The PAIgG levels were significantly elevated in CH and LC patients (mean +/- SD: 56.5 +/- 42.3 and 144.6 +/- 113.6 ng/107 cells, respectively) compared with the controls (18.9 +/- 2.5 ng/107 cells, P < 0.001 for both). Spleen volume was significantly higher only in LC (428 +/- 239) compared with CH (141 +/- 55) and control (104 +/- 50 cm3) (P < 0.001), while liver volume was not significantly different between the three groups. Correlation analyses demonstrated a significant negative correlation between platelet count with PAIgG (r = - 0.517, P < 0.001) and spleen volume (r = - 0.531, P < 0.001), and no relationship between platelet count and serum TPO level (r = 0.076). CONCLUSIONS: Serum TPO level may not be directly associated with thrombocytopenia in patients with chronic hepatitis and liver cirrhosis. In contrast, spleen volume and PAIgG are associated with thrombocytopenia in such patients, suggesting that hypersplenism and immune-mediated processes are predominant thrombocytopenic mechanisms.

The effect of splenectomy for hypersplenism on whole body protein turnover, resting metabolic rate and growth in sickle cell disease.

OBJECTIVE: To determine whether, in the same individual, an observed fall in whole body protein turnover following splenectomy in children with hypersplenism and homozygous sickle cell (SS) disease is associated with a measurable fall in resting metabolic rate (RMR) and an increase in rate of growth. SUBJECTS: Six children (5 SS disease, 1 S beta degree thalassaemia), aged 68 to 126 months, were studied before and after splenectomy for hypersplenism. DESIGN: Protein turnover was measured by the end product method using prime/intermittent oral doses of 15N-glycine and RMR by indirect calorimetry before preoperative transfusion and repeated at least eight weeks after splenectomy. Height and weight velocities were measured over six month periods before and after splenectomy. SETTING: University Hospital of the West Indies in Jamaica and the Medical Research Laboratories (Jamaica). RESULTS: After splenectomy protein turnover fell significantly by 30% and RMR by 34 kJ/kg/d. Mean weight velocity which was below normal before surgery, z = -2.3, improved significantly after surgery, z = 0.7, (P = 0.03). Height velocity increased in two children but the mean height velocity did not change following splenectomy. The reduction in protein turnover was estimated to account for 62% of the fall in RMR. CONCLUSION: This study confirms that there is a significant reduction in energy expenditure following splenectomy for hypersplenism in SS disease. A reduction in protein turnover was a major contributor to the saving in energy, although it is not clear whether it accounted for all. In the present group of children the energy saved was associated with an improvement in the wasting present before splenectomy.

The effect of splenectomy for hypersplenism on whole body protein turnover, resting metabolic rate and growth in sickle cell disease

OBJECTIVE: To determine whether, in the same individual, an observed fall in whole body protein turnover following splenectomy in children with hypersplenism and homozygous sickle cell (SS) disease is associated with a measurable fall in resting metabolic rate (RMR) and an increase in rate of growth. SUBJECTS: Six children (5 SS disease, 1 S beta degree thalassaemia), aged 68 to 126 months, were studied before and after splenectomy for hypersplenism. DESIGN: Protein turnover was measured by the end product method using prime/intermittent oral doses of 15N-glycine and RMR by indirect calorimetry before preoperative transfusion and repeated at least eight weeks after splenectomy. Height and weight velocities were measured over six month periods before and after splenectomy. SETTING: University Hospital of the West Indies in Jamaica and the Medical Research Laboratories (Jamaica). RESULTS: After splenectomy protein turnover fell significantly by 30 percent and RMR by 34 kJ/kg/d. Mean weight velocity which was below normal before surgery, z = -2.3, improved significantly after surgery, z = 0.7, (p = 0.03). Height velocity increase in two children but the mean height velocity did not change following splenectomy. The reduction in protein turnover was estimated to account for 62 percent of the fall in RMR. CONCLUSION: This study confirms that there is a significant reduction in energy expenditure following splenectomy for hypersplenism in SS disease. A reduction in protein turnover was a major contributor to the saving in energy, although it is not clear whether it accounted for all. In the present group of children the energy saved was associated with an improvement in the wasting present before splenectomy.(AU)

Correlation of splenic function with the splenic uptake rate of Tc-colloids.

The splenic uptake rate of Tc-sulphur colloid or Tc-tin colloid was measured and found to correlate well with splenic function. The normal tracer uptake rate was 0.0002/s-0.0006/s (measured uptake rate divided by measured injected activity). Lower values indicated hyposplenism (sensitivity = 0.97, specificity = 0.95), and values over 0.0006/s indicated hypersplenism (sensitivity = 0.96, specificity = 0.97). Higher values of splenic uptake were associated with proportional reductions in the white blood cell and platelet counts, and to a lesser extent the haemoglobin concentration in peripheral blood. Patients with rheumatoid arthritis had increased tracer uptake, but still below the criteria for hypersplenism, whereas patients with Felty's syndrome had tracer uptake rates in the 'hypersplenic' range.

Plasma beta-2-microglobulin and fibronectin levels in beta-thalassaemia.

The plasma beta 2-microglobulin (beta 2-MG) levels in 118 children with thalassaemia were investigated. The mean level was higher than in healthy children. A significant increase of beta 2-MG was associated with hypersplenism (3.14 +/- 0.6 mg/l). The beta 2-MG levels appeared to reflect reticuloendothelial system activity but were not related to iron overload. Fibronectin levels were generally lower than in healthy adults; profound chronic fibronectin depletion was not accompanied by an increased liability to infection.

Effect of bleeding on irradiation-induced erythropoiesis in the spleen of AKR mice.

The effect of erythropoietin, increased by bleeding, on the erythropoiesis induced by irradiation in the spleen of AKR mice, has been studied. The following parameters were measured to quantify the erythropoietic activity: the number and size of hematopoietic nodules (colonies) and proerythroblasts in the spleen, the spleen, blood and red-cell 59Fe uptake and the hematocrit and reticulocytes in the blood. Under erythropoietic stimulus an increase in the number and size of colonies was observed and these colonies were observed sooner because of their more rapid growth. The proerythroblasts in the spleen appeared earlier, and there were increases in the spleen, blood and red-cell 59Fe uptake and in the hematocrit and reticulocytes in the blood.