Effect of splenin and splenic protein-free extracts on the adenosine deaminase activity in various organs of the rat

It has been shown that the adenosine deaminase activity of intact rats increases in such lymphoid organs as the thymus and spleen under the influence of splenic protein-free extracts dissolved in the ratio of 1:100. The enzyme activity in testes does not change but its decrease (by 26.2%) is observed in the adrenal glands under the influence of the splenic protein-free extract. An analogous effect is revealed in splenin and its fractions. The splenic protein-free extract increases (by 83%) the enzyme activity in the thymus of splenectomized rats as compared to intact animals but does not change it in a homogenate of testes.     

Stimulating effect of biologically active substances from the spleen on the functional activity of hepatocytes

Studies in the effect of both splenin and spleen "protein-free extract" on the monolayer culture of chick hepatic embryos have revealed that small doses exert a stimulating effect on hepatocytes, while the large ones induce degenerative changes. Hepatotrophic characters of the "protein-free extract" are determined, while utilizing lower doses than those of splenin.     

Humoral factors of the spleen in the regulation of the calcium content in blood plasma of rats]

Introduction of protein-free peptides-enriched spleen extract increases the calcium content in blood plasma of rats. After the effect of stress factor (long swimming) it falls. The value of the index under study increases in the splenectomized rats and remains unchanged after stress and introduction of the spleen factors. The most pronounced increase of the calcium concentration is observed in the case of experimental hypersplenism induced by methylcellulose introduction. The calcium-regulating effects of introduction of the enriched protein-free spleen extract and pharmacopoeial splenin preparation are compared. It is supposed that spleen contains two humoral factors of different chemical nature which are able to influence somewhat differently the calcium content in blood plasma.     

Effect of splenic humoral factors on the cholinesterase activity of liver tissue and blood serum in experimental toxic hepatitis in rats

Activity of cholinesterase in the liver of rats with experimental toxic hepatitis decreases, while in the serum it remains unchanged. Introduction of splenin, a spleen preparation, normalizes indices of the cholinesterase activity, extract of muscles possessing no such property. Boiling-heated spleen preparation looses its activity. Activity of the studied enzyme remains unchanged both after splenectomy and splenin introduction to intact animals.     

Role of the spleen in the regulation of the calcium content of the blood plasma in health and in stress

Splenectomy is found to be followed by an increase of the calcium level in blood. Administration of the spleen extract (splenin) decreases the calcium concentration in blood plasma of splenectomized animals. The normalizing splenin effect is observed in the presence of stress when the concentration of the studied trace element sharply increases as well. It is supposed that the humoral spleen factors participate in maintenance of the constant calcium level in blood preventing its elimination from tissues.     

Immunoassay for bovine serum thymopoietin: discrimination from splenin by monoclonal antibodies

A polyclonal rabbit anti-bovine thymopoietin antiserum was used to develop a radioimmunoassay and sandwich enzyme-linked immunosorbent (ELISA) assay for the thymic hormone thymopoietin. Both assays showed slightly less sensitivity for the closely related splenic hormone splenin (SP) than thymopoietin (TP) and markedly less sensitivity for the human as compared with the bovine polypeptides. A number of murine monoclonal antibodies specific for bovine thymopoietin were generated; they were unreactive with bovine splenin and were also unreactive with human thymopoietin and splenin. A sandwich ELISA using these monoclonal anti-TP antibodies together with polyclonal rabbit anti-TP was specific for bovine thymopoietin and measured 300-500 ng/ml thymopoietin in bovine serum. Similar approaches are being pursued to develop an immunoassay for thymopoietin in human serum.     

Role of splenin and its active factor in regulating liver monooxygenase system in experimental immunodeficiency and hepatosohepatitis

An influence of splenin and its non-peptide factor of splenin (NFS) on the state of cytochrome P-450 dependent monooxygenase system (MOS) of liver microsomes in healthy animals under immunodeficiency (splenectomy, administration of gamma-aminobutyric acid (GABA) and toxic hepatosohepatitis was studied. The stimulating action of splenin and NFS on cytochrome P-450 content and MOS activity of liver microsomes in healthy animals has been established. The indices studied markedly decreased after splenectomy. The splenin or NFS administrations promote the recovery of these indices up to starting level in asplenic animals. A decrease in thymic mass dependent in GABA administration is prevented by NFS pretreatment of animals; there is no any effect of mediator acid on cytochrome P-450 content and MOS activity was noted. The preliminary administration NFS potentiates hepatotoxic effect of carbon tetrachloride and increases its inhibitory effect on P-450 dependent MOS of liver microsomes. Under the NFS action the effect in activity of the last is caused by the factor influence on the reparative processes in the liver.     

Enhanced phosphorylation of a nucleolar 110-kDa protein in rat liver by dietary manipulation

RNA polymerase 1 activity and nucleolar volume have been reported to increase in hepatocytes from rats fed a protein-free diet. Phosphorylation in vitro of a 110-kDa protein was enhanced in nuclei and nucleoli from livers of rats fed a protein-free diet. In nuclear extracts the 110-kDa protein in heat-treated nuclei was much more phosphorylated than from control liver. In contrast, casein kinase activity in the nuclear extract from control liver was comparable to that from livers of rats fed a protein-free diet. Nuclear extracts from control rat liver and livers of rats fed a protein-free diet were fractionated by DEAE-cellulose column chromatography. Casein kinase II (NII) eluted at around 0.17 M NaCl scarcely phosphorylates the 110-kDa protein. Chromatography of the nuclear extract from livers of rats fed a protein-free diet, but not from control liver, yielded fractions which eluted at 0.21-0.25 M NaCl and predominantly phosphorylated the 110-kDa protein. The phosphorylation of 110-kDa protein was not appreciably affected by a heparin concentration of 5 micrograms/ml, which completely inhibited casein kinase II. In addition, phosphorylation of the 110-kDa protein in liver nucleoli from rats fed a protein-free diet showed a lower sensitivity to heparin than that in control rat liver nucleoli. These results suggest that enhanced phosphorylation of the nuclear 110-kDa protein in livers from rats fed a protein-free diet is due to the induction of a 110-kDa protein kinase distinct from casein kinase II.     

Immunomodulatory activity of boswellic acids of Boswellia serrata Roxb

Extract of gum resin of B. serrata containing 60% acetyl 11-keto beta boswellic acid (AKBA) along with other constituents such as 11-keto beta-boswellic acid (KBA), acetyl beta-boswellic acid and beta-boswellic acid has been evaluated for antianaphylactic and mast cell stabilizing activity using passive paw anaphylaxis and compound 48/80 induced degranulation of mast cell methods. The extract inhibited the passive paw anaphylaxis reaction in rats in dose-dependant manner (20, 40 and 80 mg/kg, po). However, the standard dexamethasone (0.27 mg/kg, po) revealed maximum inhibition of edema as compared to the extract. A significant inhibition in the compound 48/80 induced degranulation of mast cells in dose-dependant manner (20, 40 and 80 mg/kg, po) was observed thus showing mast cell stabilizing activity. The standard disodium cromoglycate (50 mg/kg, ip) was found to demonstrate maximum per cent protection against degranulation as compared to the extract containing 60% AKBA. The results suggest promising antianaphylactic and mast cell stabilizing activity of the extract.     

Roles of various growth factors in growth of human osteosarcoma cells which can grow in protein-free medium.

The human osteosarcoma cell line (OST-1-PF) can grow in protein-free Coon's modified Ham's F12 medium. Growth of the cells in protein-free medium was partially density-dependent and partially depressed by medium change. An extract and conditioned medium of OST-1-PF cells contained high mitogenic activity for BALB/c3T3 cells. The growth factor in the cells was purified and identified as a basic fibroblast growth factor (bFGF)--like factor on the basis of its elution profile on heparin-affinity chromatography and the result of immunoblotting. An unidentified factor in a conditioned medium eliciting most of the DNA synthesis-stimulating activity showed a weak affinity for heparin. Various additions, including serum and growth factors, stimulated the growth of OST-1-PF cells in protein-free medium. Of these factors, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF) and bFGF were the most potent mitogens. High-affinity receptors of EGF and FGF were found on the surface of these cells. These results indicate that autonomous growth of OST-1-PF cells in protein-free medium is mainly controlled by an intracellular mechanism.     

Effect of a prolonged protein-free diet on cell size, protein synthesis and degradation of rat liver

Rats were fed a protein-free diet. After 9 weeks the animals' weight decreased to about 50% of the original. The liver weight was also decreased to about half, and most interestingly the average size of the liver cells was reduced about 50%. Liver protein synthesis was approximately 75% of controls tested in an "in vitro" system. Polysomes were found disaggregated in livers of rats on protein-free diet. This was not due to a reduced content or translatability of mRNA. eIF-2 partially purified from livers of rats on protein-free diet had the same activity as that from controls. The decrease of ATP, ADP and AMP in livers of rats on protein-free diet (19%, 42% and 58% respectively) may be responsible for the decreased rate of initiation of protein synthesis. Proteolysis in liver cytosol from rats on protein-free diet was 50% higher than in controls mostly due to lysosomal proteolysis.     

Effect of the H1-histamine-like agonist material extracted from bovine spleen on 3': 5'-cyclic nucleotides content in guinea-pig ileal smooth muscle

The effect of a water-soluble "histamine-free" splenic material that mimics the H1-receptor mediated contractile action of histamine on the guinea-pig ileum has been studied upon concentrations of cGMP and cAMP in slices of this smooth muscle preparation. The splenic extract induced a rapid and sustained decrease in the concentration of cGMP accompanied by a slow decrease in cAMP content in the ileal tissue. These results indicate that the smooth muscle-stimulating agent in splenic extract had no increasing effect on cGMP content as could be expected from the hypothesis that cGMP and cAMP might mediate the smooth muscle contraction and relaxation respectively. The data are not compatible with the general hypothesis that the action of histamine and histamine-like agonists on H1-receptors is associated with an increased concentration of cGMP.     

Role of mast cells as a trigger of inflammation in Helicobacter pylori infection.

Helicobacter pylori (H. pylori) induces severe inflammation and plays a key role in gastric mucosal diseases. In general, mast cells have been believed to play an important role in inflammation. Although mast cells were detected in the gastric mucosa, the role of mast cells in the gastric mucosal inflammation caused by H. pylori is still unclear. Therefore, we examined the effects of H. pylori water extract on the degranulation of mast cells to clarify the role of these cells in gastric mucosal inflammation induced by H. pylori. Mast cells prepared from rat abdominal cavity were incubated with H. pylori for 30 min. The protein concentrations of H. pylori water extract used in this study were 0.5-3 mg/ml. The degranulation of mast cells were monitored morphologically by phase contrast microscopy equipped with time-lapse video recording system and biochemically by measuring histamine and beta-hexosaminidase. H. pylori water extract induced the degranulation of mast cells dose-dependently. The identical experiment was performed without extracellular calcium, and no significant degranulation was found. The data indicates that the degranulation of mast cells by H. pylori water extract depend on extracellular calcium. The present results indicate that H. pylori might be involved in the gastric mucosal inflammation as a trigger of mast cell degranulation for releasing chemical mediators.     

Cellular proliferation/cytotoxicity and antimicrobial potentials of green synthesized silver nanoparticles (AgNPs) using Juniperus procera

Juniperus spp. are used as medicinal plants in many countries like Bosnia, Lebanon, and Turkey. In folk medicines, these plants have been used for treating skin and respiratory tract diseases, urinary problems, rheumatism and gall bladder stones. The objectives of this work were to synthesize silver nanoparticles (AgNPs) using a coniferous tree, Juniperus procera leaf extract and testing the synthesized AgNPs for its antimicrobial potentials, hemolytic activity, toxicity and the proliferative effects against normal and activated rat splenic cells. Leaf extract was prepared using acetone and ethanol as solvents. AgNPs were prepared using the acetone extract. AgNPs were validated using UV–Vis spectroscopy and scanning electron microscopy (SEM). Functional groups in the extract were identified using Fourier Transform Infrared (FT-IR) spectroscopy. SEM images of AgNPs showed spherical and cubic shapes with a uniform size distribution with an average size of 30–90 nm. FT-IR spectroscopy showed the presence of many functional groups in the plant extract. AgNPs showed promising antimicrobial activity against tested bacteria and fungus. AgNPs also expressed a stimulating activity towards the rat splenic cells in a dose dependent manner. Acetone as solvent was safer on cells than ethanol. Green synthesized AgNPs using J. procera might be used as a broad-spectrum therapeutic agent against microorganisms and as an immunostimulant agent.     

Polygoni cuspidati radix inhibits the activation of Syk kinase in mast cells for antiallergic activity

The antiallergic activity of Polygoni cuspidati radix (PR) and the mechanism of action by which it functions were investigated in this study. The extract of PR exhibited potent inhibitory activity in mast cells; its IC50 values were 62 +/- 2.1 microg/ml for RBL-2H3 mast cells and 46 +/- 3.2 microg/m for bone marrow-derived mast cells by antigen stimulation, and it also suppressed the expression of tumor necrosis factor-alpha and interleukin-4 in RBL-2H3 cells. According to the in vivo animal allergy model, it inhibited a local allergic reaction, passive cutaneous anaphylaxis, in a dose-dependent manner. With regard to its mechanism of action, PR inhibited the activating phosphorylation of Syk, a key signaling protein for the activation of mast cells. It also suppressed Akt and the mitogen-activated protein kinases ERK1/2, p38, and JNK, which are critical for the production of various inflammatory cytokines in mast cells. The results of the study indicate that the antiallergic activity of PR is mediated through the inhibition of histamine release and allergic cytokine production by the inhibition of Syk activating phosphorylation in mast cells.     

A chemotactic factor for rat thymocytes may regulate T-lymphocyte migration toward the thymic microenvironment

Using a modified Boyden chamber assay, extracts or culture supernatants of rat thymic stromal cells, or thymocytes were examined by chemotactic activity to rat leukocytes. Rat thymocytes responded chemotactically to the aqueous extract as well as to culture supernatants of thymic stromal cells. However, neither the extract and culture medium from concanavalin A-stimulated thymocytes nor any component of rat serum has shown such an activity. The thymic extract was fractionated into three molecular species with chemotactic activity for thymocytes. The thymocyte chemotactic factor(s) (TCFs) in the extract was distinct from known lymphocytic chemotactic factors, such as interleukin-1 (IL-1), IL-2, C5a, and the culture supernatant of stimulated thymocytes. In vitro, TCFs could attract, in addition to thymocytes, bone marrow cells, fetal liver cells, and nylon-wool nonadherent lymphocytes from peripheral blood and spleen. Lymph node cells, neutrophils, macrophages, and B cells from peripheral blood could not respond to TCFs. Thymocytes also responded to the extract of splenic stromal cells. Unlike the thymic extract, however, the splenic extract was chemotactically active for lymphocytes from lymph nodes but not for bone marrow cells. These results indicate that thymic stromal cells secrete a chemotactic factor(s) for a relatively immature type of T-lineage cells, which may by a thymus-homing progenitor T cell, while spleen may contain an attractant for a relatively mature type of T-lineage cells.     

Inhibitory effect of Pterocarpus indicus Willd water extract on IgE/Ag-induced mast cell and atopic dermatitis-like mouse models

Pterocarpus indicus Willd has been widely used as a traditional medicine to treat edema, cancer, and hyperlipidemia, but its antiallergic properties and underlying mechanisms have not yet been studied. The purpose of this study was to evaluate the antiallergic activity of Pterocarpus indicus Willd water extract (PIW) using activated mast cells and an atopic dermatitis (AD)-like mouse model. PIW decreased IgE/Ag-induced mast cell degranulation and the phosphorylation of Syk and downstream signaling molecules such as PLC-γ, Akt, Erk 1/2, JNK compared to stimulated mast cells. In DNCB-induced AD-like mice, PIW reduced IgE level in serum, as well as AD-associated scratching behavior and skin severity score. These results indicate that PIW inhibits the allergic response by reducing mast cell activation and may have clinical potential as an antiallergic agent for disorders such as AD.     

Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity

Interleukin 3 (IL 3) was initially defined as a factor in conditioned media from concanavalin A-stimulated lymphocytes (Con A CM) that induces the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of nu/nu splenic lymphocytes. To determine the spectrum of additional "biologic" activities, IL 3 was purified to homogeneity and its properties were assessed. The protein preparation was judged to be homogeneous IL 3 by the following criteria: 1) elution of a peak of IL 3 with a constant specific activity in the last step of purification, 2) presence of a single protein by SDS-PAGE analysis, 3) receptor-binding activity against IL 3-dependent cell lines, 4) a specific activity of congruent to 0.2 ng/ml required for 50% of maximal biologic activity, and 5) the presence of a single amino terminal sequence. With the use of this preparation of IL 3, the dose-response curves for 20 alpha SDH induction were identical or similar to the dose-response curves for the activities of 1) WEHI-3 growth factor, 2) mast cell growth factor, 3) P cell-stimulating factor, and 4) histamine-producing cell-stimulating factor. In addition, homogeneous IL 3 had colony-stimulating factor activity, although only approximately 2% of the total CSF activity found in Con A CM was associated with IL 3. The major peak of CSF activity could be resolved from IL 3 by DEAE column chromatography and lacked many of the biologic activities associated with IL 3.     

The fine structural localization of endogenous and exogenous peroxidase activity in human bone marrow mast cells under pathological conditions

We have examined the ultrastructural characteristics of peroxidase activity in human bone marrow mast cells. These studies were performed in three patients with systemic mast cell disease, and in another six patients showing bone marrow mast cell hyperplasia. Endogenous peroxidase activity was localized in the perinuclear cisternae and strands of endoplasmic reticulum, but never in the granules. We have also demonstrated the "in vivo" existence of exogenous peroxidase activity in two of the three cases of systemic mast cell disease. The peroxidase internalization involved its binding to the plasma membrane, followed by its incorporation into the cell by a general endocytic process comprising the uptake of dispersed peroxidase-positive material mainly by phagocytosis of granular structures containing peroxidase. The exogenous peroxidase appeared in non-membrane bound granules, vacuoles or aggregates, but we have never seen the enzyme linked to the mast cell granules.