Immunovac-VP-4, immunomodulator decreases immunosuppression and enhances the cytotoxic activity induced by the cytostatic Cisplatin

The influence of the vaccine Immunovac-VP-4, prepared from the antigens of opportunistic microorganisms, on the proliferative and cytotoxic activity on peripheral blood mononuclears (PBMN) from healthy donors in vitro and on spleen cells of CBA mice in vivo during their incubation with Cisplatin was studied. VP-4 produced a dose-dependent, stimulating effect on the proliferative potential of PBMN and, when used in the highest of all tested doses (20 microg/ml), increased the Cisplatin-suppressed proliferative activity of PBMN in 9.4-fold. VP-4 increased the cytotoxic activity of PBMN on tumor line cells K-562 (38,4 to 60.1%) and increased the cytotoxic effect of Cisplatin (68.18 to 87.56%). A single injection of VP-4 to mice stimulated the proliferative activity of spleen cells, studied ex vivo, units and partially restored their cytostatic-suppressed activity. The cytotoxic action of the spleen cells of immunized mice on tumor line cells YAC-1 was twice as great as that of spleen cells taken from intact animals and potentiated the cytotoxic action of Cisplatin. The mechanism of increasing the proliferative activity and cytotoxic effect of monomuclears under the influence of vaccine VP-4 is seemingly linked with the synthesis of cytokines, influencing the lymphokine-activated cytotoxicity of lymphocytes.     

Effect of the lymphocytosis-stimulating factor from Bordetella pertussis on murine lymphoid and hemopoietic cells

Effect of B. pertussis lymphocytosis-promoting factor (LPF) on the lympho-hematopoietic system of mice was studied. The injection of LPF was shown to sharply enhance endogenous colony formation and to induce a severe depletion of thymus cells, reaching its maximum of day 4. Thymocytes obtained on day 2 or 3 after the injection of LPF produced a suppressive effect on endogenous colony formation. The proliferative activity of hematopoietic stem cells sharply increased under the influence of LPF, though it had no radioprotective action. On the following day after the injection of LPF a steep rise in the number of hematopoietic stem cells was observed in the blood of mice: their content increased 20-fold in comparison with the control level. These data may be important for the evaluation of the side effects of pertussis vaccine on the lympho-hematopoietic system.     

Effect of thyroxine and prednisolone and the exogenous RNA induced by them on the proliferation of cells

As demonstrate experiments performed on cells of the primary culture of the newborn rat kidney, injection of thyroxin stimulates, and addition of prednisolone inhibits the proliferative activity of the cells in the culture. At a combined and simultaneous administration of these two hormones, as well as at injection of thyroxin 2 h before prednisolone, the thyroxin program, stimulating the cell proliferation is expressed. When thyroxin is administered 2 h after prednisolone, during first 12 h the prednisolone program is expressed, and then the proliferative activity of the culture returns to the initial level. At the cooperative action of oppositely directed factors for realization of the cell proliferative program, an essential role play the time factor and lag-period duration for each of the hormones interacting. Exogenic RNAs, obtained from the kidneys of the rats, to whom thyroxin or prednisolone have been injected, when they (RNAs) are injected into the culture, they produce effects, similar to those that are noted at injecting these hormones into the incubation medium. Thus, injection of the hormones to rats, results in formation, by the cells of the organ, induced RNAs, capable to transfer a hormonal signal and produce a hormone-like effect at regulation of the proliferative activity of the cell culture in the obtained exogenic RNA from the organ-donor.     

Proliferative processes in the epithelium of the autotransplant and surrounding skin of mice]

Proliferative processes were studied in epithelial cells of skin autografts and the surrounding skin in mice of CBA line. In the animals subjected to the operation but without cortisone injection (Fig. 1, a and Fig. 2, delta), a high proliferative activity in the skin epithelial cells surrounding the autograft was observed during the whole course of the experiment. In the cells of the autograft, the mitotic activity, after inhibition, restored quickly up to the original level, and in 10 days it sharply increased and did not differ from that in the epithelium of the surrounding skin. The amount of DNA-synthesising cells in the epithlium of the autograft at first remained at the original level, and then, after a short sharp rise, it decreased up to its level in the surrounding skin and then was changing nearly in the similar fashion with it. After repeated injection of cortisone both indices of proliferative activity (Fig 1, delta and Fig. 2, delta) in the epithlium of the autograft and of the surrounding skin gradually increased although slower than in the controls.     

Effect of an extract of mouse Ehrlich ascitic tumor cells on mitotic activity and DNA synthesis in that tumor]

The extract of Ehrlich's ascitis tumour cells depressed specifically the proliferative activity of this tumour cells. This was expressed in a marked reduction in the number of dividing and DNA-synthesizing cells after the extract injection. The mitotic index fell considerably as soon as 2 hours after the injection, reached the minimum in 4 to 5 hours and was restored to the control level in 9 to 12 hours. The radioactive index appeared to be evenly decreased in the course of 18 hours of the experiment.     

Proliferative activity of the surface foveolar gastric epithelium in acetysalicylic acid injury

The proliferative activity of gastric mucous cells (GMC) was studied in white mice following the administration of acetylsalicylic acid (200 mg/kg). One hour before sacrifice all the animals received intraperitoneal injection of 3H-thymidine. The mitotic index and index of labeling nuclei were calculated by means of radioautographers 3, 10 and 20 days after administering the drug. Following 3-day exposure to acetylsalicylic acid the proliferative activity of GMC remained unchanged as compared to controls. Long-term administration (10 and 20 days) of the drug produced no increase in the number of erosions, but there was a statistically significant rise in the proliferative activity of GMC. This rise was accounted for by increased number of proliferating cells in the foveated and cervical divisions of the glands as well as due to the extension of the zone of distribution of precursor cells in the gastric glands as far as the basal divisions. These changes may be considered as manifestation of the protective-adaptive reaction of the gastric mucosa in response to the damaging effect of acetylsalicylic acid.     

Proliferative effect of androst-4-ene-3,17-dione and its metabolites in the androgen-sensitive LNCaP cell line

As a therapeutic approach for the treatment of androgen-sensitive diseases, it would be tempting to lower the level of the potent androgens testosterone (T) and dihydrotestosterone (DHT) by using inhibitors of type 3 and type 5 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). However, the efficiency of such a strategy will be optimal only if androst-4-ene-3,17-dione (Delta4-dione), the precursor of T, does not possess per se agonist activity on the androgen receptor (AR). To determine if the proliferative effect previously observed on AR(+) cells for Delta4-dione originates from its direct (per se) action on AR or from its transformation into a metabolite, we started a series of experimentations using the human prostate cancer LNCaP cell line, which expresses a highly sensitive AR. By real-time RT-PCR analysis, we detected type 1 5alpha-reductase (5alpha-R), a small amount of type 5 17beta-HSD, but not type 2 5alpha-R nor type 3 17beta-HSD. We then studied the transformation of labeled Delta4-dione in LNCaP cells after 1-7 days and the most important metabolite detected was 5alpha-androstane-3,17-dione (A-dione), which is the product of 5alpha-R activity. We measured only low levels of androsterone (ADT) and epi-ADT. This result was next confirmed by using an inhibitor of 5alpha-R that completely inhibited the transformation of Delta4-dione into A-dione, and consequently into ADT and epi-ADT. The proliferative effect of Delta4-dione (carefully purified) on LNCaP (AR(+)) cells was next determined in presence or absence of the 5alpha-R inhibitor. Although the cells proliferate in the presence of Delta4-dione only, no cell proliferation was observed with a combination of Delta4-dione and 5alpha-R inhibitor, suggesting that Delta4-dione is not androgenic per se. We next determined that A-dione and epi-ADT stimulated cell growth with the same pattern and potency as Delta4-dione, whereas ADT had a 3.5-fold lower proliferative activity. In conclusion, Delta4-dione is not in itself an agonist steroid on LNCaP (AR(+)) cells, and its proliferative activity appears to be mediated by its transformation into A-dione and/or into epi-ADT.     

IL-4 (B cell stimulatory factor 1) exhibits thymocyte growth factor activity in the presence of IL-2

The proliferative activity of thymocytes cultured with IL-2 and submitogenic concentrations of PHA is increased by 3- to 10-fold in the presence of IL-4. In contrast, IL-4 alone is unable to induce proliferative activity in thymocyte cultures and its synergistic activity is only apparent to concentrations of IL-2 above 1 U/ml. The costimulatory activity of IL-4 is abrogated by the monoclonal anti-IL-4 antibody 11B11. Furthermore, potentiation of the IL-2-mediated thymocyte proliferation is not seen with IL-1, IL-3, IFN-gamma, and granulocyte-macrophage CSF. Thymocytes are at least as responsive to IL-4 as B cells and the IL-4 costimulatory activity in fractionated thymocytes appears to be restricted mainly to the Lyt-2+/L3T4- population. In contrast, purified resting mature T cells do not respond to IL-4 plus IL-2, although they did proliferate in response to IL-4 in combination with PMA. These findings indicate that thymocytes and mature T cells are responsive to the costimulatory activity of IL-4 under quite different conditions, and that IL-4 may play an important role in thymocyte maturation in the thymus.     

DNA synthesis in the thymus of the adult newt Notophthalmus viridescens.

Lymphocytic activity was examined in thymuses of adult newts by studying the number, location, morphology and fate of cells within thymuses which had been processed for autoradiography 15 min, 2 and 4 h, and 2, 4, and 10 days subsequent to the injection of tritiated thymidine. Results of this study indicate (1) that the adult thymus is a highly proliferative organ, (2) that large and medium-sized lymphocytes present in the peripheral parenchyma give rise to smaller lymphocytes which move centrally and emigrate from the thymus, and (3) that many thymocytes leave the thymus within 2-4 days after they have been produced. The significance of these findings is discussed.     

An increase in the incidence of tritium-induced leukoses with the repeated administration of a bacterial lipopolysaccharide

The incidence of leukoses upon long-term administration of tritium oxide to Wistar rats increased by 15.6% as compared to 2.1% in controls. The repeated injection of bacterial lipopolysaccharide caused a 2.3-fold increase in the occurrence of hemoblastosis induced by the radionuclide mainly due to the increase in the proliferative activity of haemopoietic precursors because of the increased demand for mature cells.     

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.     

Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland.

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using 3H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMWa) waves gave a mean Gis transit time of 21-2 hr, mean Gis +S transit time of 27-0 hr, and mean Gis + S + G2 transit time of 35-8 hr for a single injection of isoprenaline, where Gis is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the Gis phase dropped from 18-1 to 1-3 while the other variances remained unchanged. The estimated proliferative fraction was 0-24 after a single injection of isoprenaline, and 0.31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated 3H-TdR injections, were 0-21 and 0-36 respectively.     

The effect of estrogen on epithelial cell proliferation and differentiation in the crypts of the descending colon of the mouse: a radioautographic study

The regulatory role of estrogen on cell population kinetics in the descending colon was studied in intact female and ovariectomized mice. In the colonic crypts from intact mice, the crypt size (the number of epithelial cells per crypt column) and the proliferative activity of epithelial cells fluctuated slightly during the estrous cycle. Peak cellularity per crypt column was exhibted during estrus and early diestrus, whereas peaks in labeling index were seen during estrus and late metestrus. While the population size of mucous cells showed a minimal variation, the number of proliferative vacuolated cells per crypt column varied inversely with that of differentiated columnar cells during estrous cycle. The vacuolated cells were increased in number in the preovulatory phase and the columnar cells in the postovulatory phase. Three weeks after bilateral ovariectomy, the colonic crypt appeared to reach a new steady state, which was characterized by a small crypt size, a decrease in the number of differentiated cells, an increase in the relative number of proliferative cells and a relative increase in the proliferative activity of the crypt as compared to intact mice. When ovariectomized mice were treated with estrogen, the number of 3H-thymidine-labeled cells in the crypt was decreased as compared to untreated ovariectomized mice, the decrease being greater after a single injection than after multiple injections of estrogen, and the vacuolated-columnar cell line being affected more than mucous cell line. Meanwhile, the crypt size as well as the population size of differentiated cells in the crypt failed to return to normal after estrogen treatments. Thus, estrogen did not promote differentiation of epithelial cells in the crypt.     

Hetero-organic antigens of renal nature in the composition of the nuclear nonhistone proteins in the regenerating rat liver

In the nuclear non-histone proteins of the rat liver, on the 4th day after a partial hepatoectomy or hepatocarcinogen injection as well as in the hepatocellular tumour cells, some heteroorganic antigens of kidney nature (HAkid) are found and characterized immunochemically. These HAkid can be eluted at 0.4-0.5 M NaCl during gradient chromatography on phosphocellulose. They possess some proper phosphoproteinkinase activity. The appearance of HAkid in rat liver may be considered, on the one hand, as a manifestation of some malignant factor (carcinogen action, tumour cells) and, on the other hand, they are obviously connected with proliferative activity of the regenerative rat liver cells (hepatectomy effect). It is very likely that both the sides of this phenomenological effect of HAkid in rat liver are the consequence of a specific expression along the cell oncogens.     

Influence of growth hormone and thyroxine on cell kinetics in the proximal tibial growth plate of Snell dwarf mice

In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [3H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. The labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr. In untreated dwarf mice after [3H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. The number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G0 or prolonged G1 phase. Both hGH and T4 treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G2 phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T4 stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [35S]-sulphate.     

Neonatal injection of CD3 antibody into nonobese diabetic mice reduces the incidence of insulitis and diabetes

Over 80% of nonobese diabetic (NOD) mice develop lymphocytic infiltrates of their pancreatic islets (insulitis) by 6 wk of age and 50% of the females are diabetic by 6 mo of age. The incidence of insulitis in NOD mice injected once as neonates with 250 micrograms of the CD3 antibody, 145.2C11, was 8% at 10 wk of age, increasing to 25% at 32 wk of age. Fewer than 10% of these animals developed diabetes by 8 mo of age. Neonatal administration of 145.2C11 reduced the proliferative responses of spleen cells to mitogen stimulation 2 and 4 wk postinjection and expression of TCR was reduced 1 to 5 wk postinjection. The percentage of CD4 and CD8 cells in the spleen was transiently reduced after injection and the frequency of Pgp-1+-high cells (putative memory cells) was increased 2 to 4 wk postinjection, suggesting that in vivo administration of the antibody caused some T cells to divide as well as transiently reducing T cell numbers. IL-2R expression was not detected on spleen cells in the 4 wk after antibody injection. The phenotypic and functional changes after neonatal CD3 antibody injection resolved by 8 wk of age. The control and injected mice grew normally and made equivalent IgG antibody responses to injected human IgG. Neonatally injected 145.2C11 antibody was cleared from the circulation with a terminal half-life approximating to 21 days but greater than 90% of antigen binding activity was lost 6 days after injection. Protection from diabetes did not follow neonatal elimination of T cells with CD4 and CD8 antibodies, nor the injection of a TCR subset antibody, F23.1. Our data suggest that the neonatal T cell repertoire is open to modulation by a single injection of a CD3 antibody and they offer a new experimental approach to immunotherapy in an animal model of type 1 diabetes.     

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. II. Induction of helper activity demonstrated by TNP-haptenated ABA-peptide-Ficoll

In an effort to study T cell functions in Lewis rats immunized with ABA-N-acetyl-L-tyrosine (ABA-tyr), we developed an antigen that provides a sensitive assay of ABA-specific helper function that is read as an increase in TNP-specific plaque-forming cells (PFC). This antigen has ABA coupled to AECM-Ficoll by virtue of a tripeptide (tyr-ala-ala) spacer and TNP coupled to the AECM side chains. At subimmunogenic doses, this antigen induced 400 anti-TNP PFC/10(6) spleen cells in ABA-tyr-immunized rats. As many as 8000 PFC/10(6) spleen cells were induced with larger doses of antigen (200 micrograms). By contrast, only 490 PFC/10(6) spleen cells could be induced with 1 mg of the conventional doubly haptenated protein carriers such as ABA-BSA-TNP. Both direct and indirect PFC were induced by this antigen in primed rats. The use of this antigen and passive transfer techniques to study ABA-specific helper activity revealed some differences from ABA-specific delayed-type hypersensitivity (DTH) and in vitro proliferation, which were studied previously. Cells responsible for helper activity appeared sooner after immunization and were found most prominently in peritoneal exudates but also significantly in spleen where the cells responsible for DTH or in vitro proliferative responses were never found. By contrast, helper cells were not seen in lymph nodes, where some proliferative activity could be found. Of these three ABA-specific T cell functions, helper activity was least easily suppressed by the previously used regimens of ABA-tyr in incomplete freunds adjuvant (IFA). Moreover, helper activity appears after injection of ABA-tyr in IFA, a method that has never in our hands yielded detectable DTH or in vitro proliferative responses. Despite these differences, phenotyping with monoclonal antibodies indicated that cells responsible for helper and proliferative activities were both W3/25+ and OX8-.     

Age-related changes of epidermal cell kinetics in the hairless mouse

Cell kinetic variables in normal untreated hairless mice were studied in order to observe possible age-related changes. Generally, groups of 4 male and 4 female mice were subjected to study at various ages from one to 115 weeks. The number of basal and suprabasal cells per microscopic field was observed, and after injection of tritiated thymidine the mean labelling index, the average specific activity and the mean grain count were scored. After injection of Colcemid, the average number of Colcemid-arrested mitoses was counted. With flow cytometry the fraction of cells in S and in G2 + M was also observed. In general, both the number of suprabasal cells and the proliferative variables were significantly lower in the very young mice. They increased to slightly above normal values at about 20-22 weeks of age, and then fluctuated a little with two additional possible peaks at 40-50 and around 80 weeks, respectively, and two troughs some weeks after the peaks. However, this rhythmicity was slight and not significant. Thus the only significant age-related pattern was that very young mice have a thin epidermis and low proliferative variables. These values increase up to the age of 20 weeks, and from then on there are no obvious and significant alterations, only slight rhythmic undulations almost within normal limits. The importance of cell kinetic changes with age for epidermal carcinogenesis is discussed in relation to these observations.     

An ultrastructural study of proliferative nephritis induced experimentally by a monoclonal antibody against mesangial cells: replacement of mesangial cells by cells of the monocyte-macrophage system

An experimental nephritis accompanied by transient proteinuria can be produced by an intravenous injection of the monoclonal antibody, 1-22-3, raised against isolated rat glomeruli. The present study deals with the ultrastructural changes in the glomeruli in rats after the injection of this antibody. At 2 h after injection, all the mesangial cells had completely degenerated and neutrophils invaded most mesangial areas. Monocytes occupied the vacant mesangial areas at 24 h and gradually increased in number over the next 4 days. At 4 and 6 days, macrophage-like cells, possibly derived from monocytes, underwent frequent mitosis, resulting in a remarkable proliferation of these cells. The interpretation of these cells as macrophages was strongly supported by the fact that they contained previously injected latex particles in large numbers. From 2 to 4 weeks after injection, the macrophage-like cells gradually transformed into cells indistinguishable from normal mesangial cells. In the present experimental nephritis where all mesangial cells were initially destroyed, cells of the monocyte-macrophage system appear to play a leading role in the pathogenesis of the ensuing proliferative glomerulonephritis, and represent the source of the replacing mesangial cells.     

Changes in the rhythms of the alpha-fetoprotein content of the blood serum of mice with hepatoma 22A and of tumor cell proliferation depending on the time of cyclophosphane administration

A study was made of the content of alpha-fetoprotein (alpha-FP) in the blood serum and of proliferative processes in hepatoma 22A-bearing mice after injection of cyclophosphamide in a dose of 150 mg/kg at varying time of 4-day tumor growth. The data obtained indicate that the time of appearance of alpha-FP in the blood, the pattern of rhythmic fluctuations in protein content and in the number of DNA-synthesizing and mitotic cells of the tumor depend on the time of the cytostatic injection during the day. Besides, cyclophosphamide exerts a more powerful inhibitory effect on the content of alpha-FP in the blood serum and less powerful on the proliferative processes in hepatoma 22A.