Roles of adroxazine, a new heterocyclic hormone of the adrenals, and leucogenenol, a thymothyroid hormone, in the formation of blood cells

Treatment of normal rats with the thymothyroid hormone leucogenenol accelerates the rate at which the 'functional' cells, neutrophils, red blood cells and lymphocytes, develop in the bone marrow from their corresponding committed precursor cells. Following the initial treatment of normal rats with leucogenenol, there is a temporary elevation in the bone marrow of the relative concentrations of myeloblasts, rubriblasts and lymphoblasts. It has now been found that bilaterally adrenalectomized rats have a different response from normal rats to treatment with leucogenenol. Although treatment of bilaterally adrenalectomized rats with leucogenenol accelerates the rate of development of their functional cells, the initial injection of leucogenenol is followed by a temporary decrease in the relative concentrations of myeloblasts, rubriblasts and lymphoblasts in their bone marrow. Additionally, adrenalectomized rats treated concurrently with tritiated thymidine and leucogenenol show a significant lower percentage of labeled cells in their bone marrow than do correspondingly treated normal rats. These results indicate that adrenalectomized rats have a lower than normal concentration of stem cells in their bone marrow that can be committed to become functional cells. Treatment with adroxazine, a new recently isolated heterocyclic hormone of the adrenals, causes adrenalectomized rats to respond as normal rats to the injection of leucogenenol. There is a temporary elevation of myeloblasts, rubriblasts and lymphoblasts in their bone marrow as well as a normal increase in the percentage of cells that are labeled following concurrent treatment with tritiated thymidine and leucogenenol. It may be concluded that treatment with adroxazine increases the rate of replication of uncommitted bone marrow stem cells while treatment with leucogenenol increases the rate at which committed cells develop into their respective functional cells. A scheme is presented to show the suggested roles that leucogenenol and adroxazine play in regulating the formation of blood cells.     

Association of leucogenenol,a thymothyroid hormone,with carrier proteins in the thymus

Leucogenenol a heterocyclic enolic thymothyroid hormone (MW 383) whose concentration in the serum regulates the rate at which already committed cells of the bone marrow develop into functional cells, was found to be associated in the thymus with a carrier protein. The carrier protein for leucogenenol is not precipitated by heating to 80° but following this treatment leucogenenol is precipitated in association with proteins precipitated by acetone and then by saturated ammonium sulfate. On chromatography on Sephacryl G-200 it was found that leucogenenol was associated with proteins of MW approximately 38,000. Leucogenenol is not eluted from the chromatographic column if it is not associated with its carrier proteins. It is suggested that other hormones such as those associated with the reproductive cycle or compounds that result from tissue damage induce the liberation of leucogenenol from its carrier protein in the thymus to the circulation where it is associated as previously described, with a protein of approximately MW 300,000.     

Effect of leucogenenol,a thymothyroid hormone,on the enzymes and several other constituents of the serum

It has been found that rats treated with the thymothyroid hormone, leucogenenol, have significantly elevated levels of alkaline phosphatase and lactate dehydrogenase in their serum 8 hrs. later. These levels remain elevated for 24 hrs. and 12 hrs., respectively. The concentrations of creatine phosphokinase and aspartate aminotransferase as well as total protein, albumin, calcium, inorganic phosphorus, glucose, urea nitrogen, uric acid and creatinine in the serum are not affected by treatment with leucogenenol. These results are in agreement with the previously reported findings that treatment with leucogenenol increases the rate of development of blood cells of the bone marrow, increases the rate of recovery from immunosuppression and enhances the immune response.     

Studies of the action of leucogenenol on the myeloid and lymphoid tissues of the sublethally irradiated mouse

Studies of the action of leucogenenol on the peripheral leukocytes, the myeloid cells found in bone marrow, and the lymphoid cells found in the spleen of sublethally irradiated mice strongly suggest that leucogenenol stimulates the maturation and/or cellular division of cells of both the myeloid and lymphoid series. Accordingly, as indicated by the increase in the number of peripheral leukocytes, as well as the increase in the number of myeloid and lymphoid cells found in the bone marrow and spleen, mice treated with leucogenenol appear to recover more rapidly than untreated controls.     

The differentiation factor in the bone marrow and blood serum of healthy subjects and in acute leukemia

The factor of differentiation--i.e. systemic morphogen of connective tissue (SMCT)--have been discovered in bone marrow and blood serum of healthy humans. SMCT calls forth the differentiation of mesodermal cell types in early embryonic amphibian cells. These cell types are the following: notochords, muscles, mesothelium, blood cells, mesenchyme. Under the influence of the punctates of bone marrow the frequency of muscle and blood cell appearance is not constant, which might be the consequence of the individual variability of SMCT. Under the influence of bone marrow and blood serum in patients with acute lymphoblastic, monoblastic and myelomonoblastic leukemia the embryonic cells differentiate only into atypical epidermis, which proves the absence of the SMCT activity in the sources used. In some cases under the influence of bone marrow from patients having the same disease the early embryonic cells differentiate into mesodermal cell types, which normally appear under the low concentration of SMCT. This was observed however only in those cases when bone marrow or blood serum have been taken from patients in the state of remission. In patients with remission the correlation is observed between the activity of factor of differentiation in bone marrow and that of blood serum.     

Isolation of leucogenenol from bovine and human liver

A compound has been isolated from both bovine and human liver that on intravenous or intraperitoneal injection into animals induces in 4-12h an increase in the number of peripheral neutrophils, and in 12-24h an increase in the number of peripheral lymphocytes. At 24h after injection there is also a two- to three-fold increase in the relative number of myeloblasts in the bone marrow. The procedure for isolation and the physical and chemical properties identify the compound as leucogenenol, isolated from the metabolic products of Penicillium gilmanii by Rice (1966).     

Cellular events in tolerance. II. Thymus-bone marrow cell cooperation in the immune response to BSA in Wistar Furth rats

Normal bone marrow cells from Wistar Furth rats were competent to transfer immune responsiveness to bovine serum albumin to thymectomised, irradiated, syngeneic recipients. When the bone marrow cells were taken from donors thymectomised early in life they were incompetent, but competence was restored by addition of normal thymus cells. It was concluded that normal Wistar Furth bone marrow cells contain some thymus-derived cells. Thymus cells from tolerant donors were less effective in cooperation with bone marrow cells, however the thymus cells appeared less tolerant than their donors.     

Cytoplasmic immunofluorescence of bone marrow plasma cells producing immunoglobulins of the four IgG subclasses.

Immunofluorescence studies were performed on bone marrow samples from 21 individuals with normal or slightly elevated serum IgG levels. On the average, 40.7 percent of the IgG-positive plasma cells contained molecules of the subclass IgG; 30.6 percent had intracellular IgG2; 21.7 percent, IgG3, and 7.0 percent, IgG4. These figures were compared to the relative serum IgG subclass concentrations. The principal finding was the discrepancy between the high relative number of IgG-3 producing bone marrow plasma cells and the low relative IgG3-producing bone marrow plasma cells and the low relative IgG3 serum concentration (5.5 percent of the total IgG). It was explained by the short biologic half-life of molecules of this subclass.     

Romanovsky Staining of Low Viscosity Nitrocellulose Sections of Bone Marrow

In the detailed study of bone marrow, sections of undisturbed marrow are essential in determining the distribution and relative proportion of the various hemopoietic cells and the relative volume occupied by marrow cells, fat, and blood in the various portions of the same bone or of different bones. As a result of trial of a number of fixatives, stains, and embedding technics, the following technic was evolved. It gives the best combination of good differential staining with the least shrinkage and cellular distortion, and is used routinely by the author in the study of bone marrow lesions in rats.     

Effect of some stressors on the mitotic regimen of the corneal epithelium and on the level of aneuploid bone marrow cells of adrenalectomized albino rats

It has been shown that pyrogenal administration or one-hour hyporthermia at 28--30 degrees C did not induce development of reactive inhibition in adrenalectomized rats but produced a significant increase in the level of pathological mitoses in the cornea of intact (from 4.3% to 6.3%) and adrenalectomized (from 10.6% to 12.5%) rats subjected to stress. The karyotypic analysis of the bone marrow cells under such conditions showed a significant rise in the number of aneuploid (hupo- and hyperdiploid) cells.     

The transmural migration and release of blood cells in acute myelogenous leukemia.

The transmural passage of malignant blood cells from the extravascular parenchyma into sinusoidal lumen has been studied in the bone marrow of rats with myelogenous leukemia. The Shay myelogenous leukemia was chosen as a model system because an increased bone marrow cellularity is, in this leukemia, usually accompanied by an increase in circulating myeloid cells. By means of light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) it was found that the sinusoidal endothelial lining of the bone marrow remains intact and continuous even in advanced stages of the disease. SEM shows that the malignant myeloblast-like cell enters the sinusoidal lumen by means of a temporary migration pore, which appears only during the transmural passage of the cell. Certain nondegenerative changes in the sinusoidal blood vessels are associated with the myelogenous leukemia. The normal radial alignment of sinusoids about the central sinusoid is changed into a tortuous pattern, and intraluminal cytoplasmic bridges which impede the blood flow are formed by the endothelial cells.     

Chemical determination of leucogenenol and its production by Penicillium gilmanii

A method for the quantitative determination of leucogenenol, a leucocytosis-inducing compound produced by Penicillium gilmanii, has been developed. The formation of leucogenenol in P. gilmanii and its diffusion into the culture fluid has been followed over a period of 12 weeks. The highest concentration of leucogenenol was found in the mycelium of rapidly growing cultures. The greatest concentration occurred in the culture fluid, however, after cessation of rapid growth. The concentration of leucogenenol in the mycelium of cultures over 4 weeks old fluctuated widely. This fluctuation was apparently due to the formation and rapid growth of daughter colonies.     

Incorporation of 3H-2,3-DL-valine into pancreatic proteins of adrenalectomized rats stimulated with pancreozymin-CCK and secretin

Incorporation of labelled valine was investigated in bilaterally adrenalectomized rats stimulated with pancreozymin-CCK and secretin 14 days after operation. The growth of rats, the blood pressure, the amount of pancreatic juice and the amylase output was less in the adrenalectomized animals as compared with sham operated or adrenalectomized and corticosterone substituted controls. The amylase concentration in the pancreatic juice reveals no difference between the groups but the output of amylase remained below that of the controls. The incorporation of labelled valine was higher in the pancreatic tissue and proteins in the adrenalectomized animals than in the controls. It is assumed that the adrenalectomy causes primarily a depletion in the metabolic activity and systemic blood pressure; diminished pancreatic juice secretion and output of amylase are only secondary consequences which are not compensable by hormonal stimulation of pancreatic secretion.     

Effect of hydrocortisone on ACTH, growth hormone, insulin and glucose in the blood of bilaterally adrenalectomized patients

This study is aimed at elucidating the mechanism of paradoxical rise in plasma ACTH levels in response to glucocorticoids, observed by several authors in bilaterally adrenalectomized patients with Cushing's disease. Six control subjects and fourteen patients bilaterally adrenalectomized for Cushing's disease were given a dose of 200 mg hydrocortisone sodium succinate by 3-5 mm i.v. injection. Plasma ACTH (in 6 patients), serum cortisol, growth hormone (GH) and insulin and blood glucose levels were estimated at 0, 30, 60, 90, and 120 minutes. The administration of hydrocortisone significantly suppressed plasma ACTH levels only at 60 min. In one case a slight rise in ACTH level during the test was observed. A significant fall in blood glucose levels was found only in the adrenalectomized patients. No significant changes in serum insulin and GH levels were noted. The possible mechanisms are discussed, especially the potential role of transient glucose deficiency in the pathophysiology of plasma ACTH increase in response to hydrocortisone in the bilaterally adrenalectomized patients.     

Osteopetrosis in the toothless (t1) rat: presence of osteoclasts but failure to respond to parathyroid extract or to be cured by infusion of spleen or bone marrow cells from normal littermates.

Osteoclast have been observe for the first time in toothless (t1) rats, a mutation with inherits osteopetrosis as an autosomal recessive. The ability of t1 rats to raise the serum calcium concentration after injection of parathyroid extract was severely limited when compared with normal littermates. In addition, osteopetrosis in t1 rats is not cured by radiation and infusion of normal spleen or bone marrow cells from normal littermates, a method know to cure osteopetrosis in mutants of this and other species. This indirect evidence for a reduction in bone resorption as a cause of osteopetrosis in this mutation and the failure of transplanted cells to cure the disease are discussed in relation to the development and function of osteoclasts.     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

Primitive progenitor cells in the blood of patients with chronic granulocytic leukemia

We measured the number of blast colony-forming cells (Bl-CFC) in the blood of 11 patients with untreated chronic granulocytic leukemia (CGL). The culture system used detects three types of Bl-CFC (Types I, II and III) in normal marrow, of which Bl-CFC (I) are the most primitive and might represent the putative hemopoietic stem cell. The mean numbers of Bl-CFC (I) in CGL blood, normal bone marrow and normal blood were 134 +/- 29 (+/- SEM), 127 +/- 21 and 1.5 +/- 0 respectively per 1 X 10(6) mononuclear cells. These findings are consistent with the concept that CGL is due to a primary increase in stem cell numbers with secondary increases in committed progenitor and leukocyte numbers.     

Organization of haemopoietic stem cells: the generation-age hypothesis.

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 13 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

去卵巢大鼠血清和骨髓细胞碱性磷酸酶水平变化的动态观察

目的：动态观察去卵巢大鼠血清和骨髓细胞碱性磷酸酶（ ALP）水平的变化。方法将80只3月龄雌性SD大鼠按体重分层后随机分为基础组以及假手术和去卵巢3、6、12、24周组。分别在手术前（0）和手术后3、6、12、24周腹主动脉取血处死各组大鼠,分离血清,制备骨髓细胞甩片,用721分光光度计检测血清ALP水平的变化;用显微镜计数骨髓细胞甩片ALP阳性染色细胞的数目。结果在假手术组大鼠中,血清ALP水平在手术后3周显著上升并持续到手术后6周,但在手术后12周开始显著下降并持续到手术后24周;骨髓细胞ALP阳性染色细胞数目在手术后3周显著上升并持续到手术后12周,但在手术后24周却显著下降。在去卵巢组大鼠中,血清ALP水平在手术后3周显著上升,到手术后6周开始显著下降并一直持续到手术后24周;骨髓细胞ALP阳性染色细胞数目在手术后3周显著下降并持续到手术后24周。从手术后3周开始,去卵巢组大鼠血清ALP水平均显著高于假手术组大鼠,但骨髓细胞ALP阳性染色细胞数目均显著低于假手术组大鼠。结论假手术组大鼠血清和骨髓细胞ALP水平变化趋势基本相似,但去卵巢大鼠血清和骨髓细胞ALP水平变化趋势不同。     

Immunohistochemical demonstration of a CRF-associated neurophysin in the external zone of the rat median eminence

Cell and Tissue Research - In picric acid-formalin fixed, paraplast embedded median eminence sections from normal and bilaterally adrenalectomized rats, the amount of “Gomori-positive”...