Lymphocyte activation in silica-exposed workers.

Exposure to silica dust has been examined as a possible risk factor for autoimmune diseases, including systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus and ANCA-associated vasculitis. However, the underlying cellular and molecular mechanisms resulting in the increased prevalence of autoimmunity remain elusive. To clarify these mechanisms, we studied various markers of immune activation in individuals occupationally exposed to silica dust, i.e., serum levels of soluble IL-2 receptor (sIL-2R), levels of IL-2, other pro- and anti-inflammatory cytokines and lymphoproliferation. Our results demonstrate that silica-exposed individuals present important alterations in their immune response when compared to controls, as shown by increased serum sIL-2R levels, decreased production of IL-2 and increased levels of the pro-inflammatory (IFN-γ, IL-1α, TNF-α, IL-6) as well as anti-inflammatory (IL-10 and TGF-ß) cytokines. Furthermore, silica-exposed individuals presented enhanced lymphoproliferative responses. Our findings provide evidence that the maintenance of immune homeostasis may be disturbed in silica-exposed individuals, possibly resulting in autoimmune disorders.

Genetic polymorphisms and surface expression of CTLA-4 and PD-1 on T cells of silica-exposed workers.

Exposure to silica dust has been examined as a possible risk factor for autoimmune diseases, including scleroderma, rheumatoid arthritis and systemic lupus erythematosus. Since CTLA-4 [CD152] and PD-1 [CD279] are important for the maintenance of peripheral tolerance by regulating T cell responsiveness, we evaluated the expression of these molecules on the surface of CD4 and CD8 T cells, as well as single nucleotide polymorphisms (SNP) in CTLA-4 and PDCD1 genes, of 70 silica-exposed workers and 30 non-exposed, age-, ethnically- and sex-matched controls. Expression of CTLA-4 was significantly (P<0.05) reduced in CD4 T cells of exposed individuals [median=0.1% and interquartile range, IQR 0.0-0.1% (exposed), median=0.20%, IQR 0.0-0.4% (control)]. Also the expression of PD-1 was significantly (P<0.0001) reduced in both CD4 [median=0.9%, IQR 0.4-2.3% (exposed), median=5.7%, IQR 1.4-13.3% (control)] and CD8 T cells [median=0.9%, IQR 0.3-1.9% (exposed), median=5.0%, IQR 3.4-8.9% (control)]. The study of polymorphisms demonstrated a lower frequency of the A allele in the analysis of the PD1.3 SNP in the exposed group, which might be associated with the lower expression of PD-1 on the surface of CD4 T cells. Our findings provide evidence for the association of silica exposure and the maintenance of self-tolerance, i.e., the susceptibility to autoimmune disorders.

Chlorella vulgaris restores bone marrow cellularity and cytokine production in lead-exposed mice.

Chlorella vulgaris (CV) was examined for its modulating effects on the reduction induced by lead (Pb) on the numbers of marrow hematopoietic stem cells (HSCs) (c-Kit(+)Lin(-)), granulocyte-macrophage progenitors (Gr1(+)Mac1(+)) and total bone marrow cellularity. In mice gavage-treated daily with 50mg/kg dose of CV for 10 days, concomitant to a continuous offering of 1300 ppm lead acetate in drinking water, the treatment with the algae recovered the significantly reduced numbers of these cell populations to control values. As CV may have a myelostimulating effect through the induction of cytokines, we evaluated its modulating effects on the production of IL-1α, TNF-α, IFN-γ, IL-10 and IL-6. Our results demonstrated that lead significantly impairs the production of IFN-γ, IL-1α and TNF-α and increases the production of IL-10 and IL-6 and that these effects are successfully modulated by the CV treatment. The activity of NK cells, reduced in Pb-exposed animals, was raised to levels higher than those of controls in the exposed group treated with CV. Treatment with the algae also stimulated the production of IFN-γ, IL-1α, TNF-α and NK cells activity in normal mice. In addition, zinc bone concentrations, reduced in lead-exposed mice, were partially, but significantly, reversed by the treatment with CV.

Chlorella vulgaris modulates immunomyelopoietic activity and enhances the resistance of tumor-bearing mice.

We studied the effects of Chlorella vulgaris (CV) on the interaction between stromal and hematopoietic stem cells in normal and Ehrlich ascites tumor (EAT)-bearing mice. Long-term bone marrow culture (LTBMC), cytokine production, spleen mononuclear cells (SMC) proliferation (SCP), colony stimulating activity (CSA), and NK cells activity were evaluated. In tumor bearers, reduced capacity of stromal cell layer to support the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM), concomitantly to decreased numbers of total nonadherent cells in LTBMC and reduced local production of IL-6 and IL-1α, were observed. Presence of the tumor has not altered the number of stromal adherent cells. CV treatment restored the ability of stromal cells from EAT-bearing mice to produce IL-6 and IL-1α, which was consistent with increased number of nonadherent cells and higher ability to display CFU-GM in vitro. EAT growth increased SCP, serum CSA, and IL-10 production and concurrently depressed NK cell activity and the secretion of IL-2, IFN-γ, and TNF-α. Treatment of tumor-bearing mice with CV augmented CSA, SMC proliferation, NK cell activity, and the production of IL-2, IFN-γ, and TNF-α, whereas IL-10 levels where reduced. Our results suggest that CV modulates immunehematopoietic cell activity and disengages tumor-induced suppression of these responses.

Immunohematopoietic modulation by oral ß-1,3-glucan in mice infected with Listeria monocytogenes.

In this study we demonstrated that the oral administration of ß-1,3-glucan (Imunoglucan®) protects mice from a lethal dose of Listeria monocytogenes (LM) when administered prophylactically for 10 days at the doses of 150 and 300 mg/kg, with survival rates up to 40%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with LM, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Investigation of the production of colony-stimulating factors revealed an increased colony-stimulating activity (CSA) in the serum of infected mice pre-treated with Imunoglucan®. The treatment also restored the reduced ability of stromal cells to display myeloid progenitors in long-term bone marrow cultures (LTBMC) and up-regulated IL-6 and IL-1α production by these cells in the infected mice, which was consistent with higher number of non-adherent cells. Additional studies to investigate the levels of interferon-gamma (INF-γ) in the supernatant of splenocyte cultures demonstrated a further increase in the level of this cytokine in infected-treated mice, compared to infected controls. In all cases, no differences were observed between the responses of the two optimal biologically effective doses. In contrast, no significant changes were produced by the treatment with the 50mg/kg dose. In addition, no changes were observed in normal mice treated with the three doses used. All together our results suggest that orally given Imunoglucan® indirectly modulates immune activity and probably disengages Listeria induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1α, IL-6, and INF-γ).

Angelica sinensis modulates immunohematopoietic response and increases survival of mice infected with Listeria monocytogenes.

The effects of a dry extract of the roots of Angelica sinensis (Oliv.) Diels (ASE) on the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM) in normal and Listeria monocytogenes-infected mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in infected mice. Prophylactic administration of ASE (10, 25, and 50 mg/kg) stimulated marrow myelopoiesis in a dose-dependent manner and reduced spleen colony formation to control values. The dose of 50 mg/kg ASE was the optimal biologically active dose in infected mice, and this dose schedule significantly increased survival of mice infected with a lethal dose of L. monocytogenes, with survival rate up to 30%. Investigation of the production of colony-stimulating factors revealed a dose-dependent increased colony-stimulating activity in the serum of infected mice, with higher response produced by the 50 mg/kg dose. Notably, no effects were observed with the 100 mg/kg dose, compared with infected nontreated controls. Further studies to investigate the production of factors such as inteferon-γ and tumor necrosis factor-α demonstrated increased levels of both cytokines in mice infected with L. monocytogenes and treated with 50 mg/kg ASE. We propose that ASE indirectly modulates immune activity and probably disengages Listeria-induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (colony-stimulating factors, interferon-γ, and tumor necrosis factor-α).

Pre-clinical antitumour evaluation of Biphosphinic Palladacycle Complex in human leukaemia cells.

Previous studies reported by our group have introduced a new antitumoural drug called Biphosphinic Palladacycle Complex (BPC). In this paper we show that BPC causes apoptosis in leukaemia cells (HL60 and Jurkat), but not in normal human lymphocytes. IC(50) values obtained for both cell lines using the MTT and trypan blue exclusion assays 5h after BPC treatment were lower than 8.0 microM. Using metachromatic fluorophore, acridine orange, we observed that BPC elicited lysosomal rupture of leukaemic cells. Furthermore, BPC triggered caspase-3 and caspase-6 activation and apoptosis in cell lines, inducing chromatin condensation, apoptotic bodies, and DNA fragmentation. Interestingly, the lysosomal cathepsin B inhibitor CA074 markedly decreased BPC-induced caspase-3 and caspase-6 activation as well as cell death. Lysosomal BPC-induced membrane destabilisation was not dependent on reactive oxygen species generation, which was consistent with the absence of cellular HL60 and Jurkat membrane lipid peroxidation. We conclude that, following BPC treatment, lysosomal membrane rupture precedes cell death and the apoptotic signalling pathway is initiated by the release of cathepsin B in the cytoplasm of leukaemia cells. As no toxic effects for human lymphocytes were observed, we suggest that BPC is more selective for transformed cells, mainly due to their exacerbated lysosome expression.

Chlorella vulgaris up-modulation of myelossupression induced by lead: the role of stromal cells.

In this study, Chlorella vulgaris (CV) was examined for its chelating effects on the ability of bone marrow stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice, using the long-term bone marrow culture (LTBMC). In addition, the levels of interleukin (IL)-6, an important hematopoietic stimulator, as well as the numbers of adherent and non-adherent cells were also investigated. Mice were gavage treated daily with a single 50mg/kg dose of CV for 10 days, concomitant to continuous offering of 1300ppm lead acetate in drinking water. We found that CV up-modulates the reduced ability of stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice and restores both the reduced number of non-adherent cells and the ability of stromal cells from these mice to produce IL-6. Monitoring of lead poisoning demonstrated that CV treatment significantly reduced lead levels in blood and tissues, completely restored the normal hepatic ALA levels, decreased the abnormally high plasma ALA and partly recovered the liver capacity to produce porphyrins. These findings provide evidence for a beneficial use of CV for combination or alternative chelating therapy to protect the host from the damage induced by lead poisoning.

Hematopoietic response of rats exposed to the impact of an acute psychophysiological stressor on responsiveness to an in vivo challenge with Listeria monocytogenes: modulation by Chlorella vulgaris prophylactic treatment.

In this study, we investigated the hematopoietic response of rats pretreated with CV and exposed to the impact of acute escapable, inescapable or psychogenical stress on responsiveness to an in vivo challenge with Listeria monocytogenes. No consistent changes were observed after exposure to escapable footshock. Conversely, the impact of uncontrollable stress (inescapable and psychogenical) was manifested by an early onset and increased severity and duration of myelossuppression produced by the infection. Small size CFU-GM colonies and increased numbers of clusters were observed, concurrently to a greater expansion in the more mature population of bone marrow granulocytes. No differences were observed between the responses of both uncontrollable stress regimens. CV prevented the myelossuppression caused by stress/infection due to increased numbers of CFU-GM in the bone marrow. Colonies of cells tightly packed, with a very condensed nucleus; in association with a greater expansion in the more immature population of bone marrow granulocytes were observed. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected/stressed rats treated with the algae. CV treatment restored/enhanced the changes produced by stress/infection in total and differential bone marrow and peripheral cells counts. Further studies demonstrated that INF-gamma is significantly reduced, whereas IL-10 is significantly increased after exposure to uncontrollable stress. Treatment with CV significantly increased INF-gamma levels and diminished the levels of IL-10. Uncontrollable stress reduced the protection afforded by CV to a lethal dose of L. monocytogenes, with survival rates being reduced from (50%) in infected rats to 20% in infected/stressed rats. All together, our results suggest Chlorella treatment as an effective tool for the prophylaxis of post-stress myelossupression, including the detrimental effect of stress on the course and outcome of infections.

Comparative studies of the effects of Tabebuia avellanedae bark extract and beta-lapachone on the hematopoietic response of tumour-bearing mice.

The effects of Tabebuia avellanedae (TACE), traditionally prescribed in the treatment of cancer, and the naphtoquinone beta-lapachone (beta-lap) on the growth and differentiation of granulocyte and macrophage progenitor cells (CFU-GM) were studied in Ehrlich ascites tumour-bearing mice. Myelosuppression concomitant with increases in spleen CFU-GM and in serum colony-stimulating activity (CSA) were observed in these animals. Treatment with TACE (30-500 mg/kg) and beta-lap (1-5mg/kg) reversed these effects in a dose-dependent manner. The optimal biologically active doses of 120 mg/kg TACE and 1mg/kg beta-lap prolonged life span of tumour-bearing mice, both producing the same rate of extension in the duration of survival. Toxic manifestations were produced by the higher doses of beta-lap in normal and tumour-bearing mice. In spite of similarities between treatments, TACE concentrations used to treat the animals presented no traces of beta-lap, as measured by TLC and HPLC analyses. Our findings suggest that the antitumour effect of TACE and beta-lap, acting synergistically with other factors, such as specific cytokines, may result from enhanced macrophage activation against tumour cells. In addition, it is clear from our results that hematopoietic disorders produced by tumours are an important pathological condition that must be considered in drug development.

Prophylactic administration of Withania somnifera extract increases host resistance in Listeria monocytogenes infected mice.

In this study, we demonstrated that Withania somnifera L. extract (WSE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 100, 250 and 500 mg/kg for 10 days, with survival rates up to 30%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Investigation of the production of colony-stimulating factors (CSFs) revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with WSE. Further studies to investigate the levels of interferon-gamma (INF-gamma) and lymphocyte cell proliferation were undertaken. We observed dose-dependent increases in cell proliferation and in the levels of INF-gamma in mice infected with L. monocytogenes and treated with WSE. All together, our results suggest that WSE indirectly modulates immune activity and probably disengages Listeria-induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow, proliferation of lymphocytes and increased INF-gamma levels.

Antitumour activity of [1,2-di(cyclopentadienyl)-1,2-di(p-N,N-dimethylaminophenyl)-ethanediyl] titanium dichloride in xenografted Ehrlich's ascites tumour.

The effects of a new titanocene compound with an ansa ligand in the cyclopentadienyl rings, the 1,2-di(cyclopentadienyl)-1,2-di(p-N,N-dimethylaminophenyl)-ethanediyl] titanium dichloride (TITANOCENE X), on the growth and differentiation of granulocyte-macrophage progenitor cells [colony-forming unit-granulocyte-macrophage (CFU-GM)] and Natural killer (NK) cell activity in Ehrlich's ascites tumour (EAT)-bearing mice were studied. Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with TITANOCENE X (2.5-50 mg/kg/day) produced an increase in myelopoiesis, in a dose-dependent manner, and reduced spleen colony formation. In addition, the treatment of EAT-bearing mice with 3 doses of 20 or 50 mg/kg TITANOCENE X restored to normal values the reduced Natural killer cell function observed during tumour growth. In parallel, TITANOCENE X prolonged, in a dose-dependent manner, the survival of mice inoculated with Ehrlich's ascites tumour. The highest dose of 50 mg/kg prolonged in 50% the survival time of EAT-bearing mice, compared to non-treated tumour-bearing controls. In comparison with previous results from our laboratory addressing the effects of titanocenes on haematopoiesis, we observed with TITANOCENE X a similar effective profile as for bis(cyclopentadienyl) dithiocyanate titanium(IV), being both less effective than di(cyclopentadienyl) dichloro titanium(IV), since the latter not only prolonged, but also increased the rate of survival. These differences in efficacy may be due to the nature of the ansa-cyclopentadienyl ligand used in TITANOCENE X, since the C2 bridge between the two cyclopentadienyl groups will increase the hydrolytic stability by an organometallic chelate effect. Also, the introduction of two dimethylamino substituents increases the water solubility of TITANOCENE X when compared to titanocene dichloride itself.

Euphorbia tirucalli L. modulates myelopoiesis and enhances the resistance of tumour-bearing mice.

Myelosuppression concomitant with increased numbers of spleen CFU-GM was observed in tumour-bearing mice. Treatment of these animals with Euphorbia tirucalli L. (ET) (125, 250 and 500 mg/kg) stimulated marrow myelopoiesis and reduced spleen colony formation, with no differences observed between the effects of the three doses. The changes produced by the tumour in total and differential marrow cell counts were restored by the treatment with ET. Prostaglandin E2 (PGE2) levels, which were dramatically increased in tumour bearers, was also abrogated by the treatment with the plant extract. Euphorbia tirucalli L. significantly enhanced survival and concurrently reduced tumour growth in the peritoneal cavity. We propose that the modulatory effect of Euphorbia tirucalli L. on myelopoietic response and on the levels of PGE2 may be related to its antitumour activity as a possible mechanism for the regulation of granulocyte and macrophage production and expression of functional activities.

Uncaria tomentosa extract increases the number of myeloid progenitor cells in the bone marrow of mice infected with Listeria monocytogenes.

In this study, we demonstrated that Uncaria tomentosa extract (UTE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 50, 100, 150 and 200 mg/kg for 7 days, with survival rates up to 35%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Non-infected mice treated with 100 mg/kg UTE also presented higher numbers of CFU-GM in the bone marrow than the controls. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with UTE. Moreover, stimulation of myelopoiesis and CSA occurred in a dose-dependent manner, a plateaux being reached with the dose of 100 mg/kg. Further studies to investigate the levels of factors such as IL-1 and IL-6 were undertaken. We observed increases in the levels of IL-1 and IL-6 in mice infected with L. monocytogenes and treated with 100 mg/kg of UTE. White blood cells (WBC) and differential counting were also performed, and our results demonstrated no significant changes in these data, when infected mice were pre-treated with 100 mg/kg of UTE. All together, our results suggest that UTE indirectly modulates immune activity and probably disengages Listeria-induced supression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1 and IL-6).

Bone marrow progenitor cells from chemically exposed workers display an intrinsic ability for autonomous proliferation.

In this study, the autonomous proliferation of bone marrow progenitor cells (CFU-C), a pathological phenomenon observed in many hematological abnormalities, was investigated in 31 individuals who had been diagnosed as having neutropenia. Of these subjects, 18 had been chronically exposed (range of exposure 5-30 years) to a variety of petroleum distillates. We observed that the group of exposed individuals presented higher numbers of autonomous CFU-C when compared with those unexposed subjects. In addition, follow-up data demonstrated that 20% of the exposed population (4 of the 18) developed malignant hematological diseases. The autonomous CFU-C obtained from all individuals studied was composed predominantly of macrophages. This suggests an involvement of these cells in the development of hematological abnormalities, probably as a result of increased production of chemical myelotoxic metabolites.

Effects produced by Royal Jelly on haematopoiesis: relation with host resistance against Ehrlich ascites tumour challenge.

Royal jelly (RJ) was shown to exhibit immunomodulatory properties, although its biological activity is still unclear. In order to elucidate the mechanism whereby RJ activates the immunological system, we examined the role of this substance on the haematopoietic response of Ehrlich ascites tumour (EAT)-bearing mice. Our results demonstrated that RJ prevented the myelosupression induced by the temporal evolution of the tumour and abrogated the splenic haematopoiesis observed in EAT-bearing mice. The stimulating effect of RJ was also observed in vitro on the multipotent bone marrow stem cells, evaluated by the long-term bone marrow cultures (LTBMCs). The study of survival clearly showed the antitumour activity of RJ. Treatment was given prophylactically for 20 days and therapeutically for 3, 8 and 13 days. Except for the treatment with the lower dose of 500 mg/kg, given for 23 days, all the other dose schedules were able to prolong survival. A more effective antitumoural response was observed with the more prolonged treatment regimen. In this regard, the administration of RJ for 33 days produced the highest protection reaching an extension of survival at about 38%, 71% and 85% for the doses of 500, 1000 and 1500 mg/kg, respectively, whereas with the 23 and 28 days treatment schedules, survival increased at a rate of 19% and 23%, respectively, and comparable results were found among the effective doses of RJ. Increased survival rate might be related to the decreased Prostaglandin E2 (PGE2) levels observed in EAT-bearing mice after RJ treatment. These results point to RJ as a promising modifier of biological response leading to myeloprotection and antitumour activity.

Myelopoietic response in mice exposed to acute cold/restraint stress: modulation by Chlorella vulgaris prophylactic treatment.

In this study, hematopoietic cells from mice pretreated with CVE and exposed to acute cold/restraint stress were stimulated in the presence of growth factors to form colonies, thus providing accurate information about the modulation of the green algae of the stress-induced changes in the hematopoietic response. Our results demonstrated that exposure to acute stress affected hematopoiesis. Mice exposed for a 2.5-hour time period of cold and restraint presented diminished clonal capacity for CFU-GM content per femur, which was decreased by as much as 50% compared with that in control mice, in spite of the significant increase in serum colony-stimulating activity (CSA). Treatment with 50 mg/kg CVE for 5 days, previously to the stress regimen, attenuates the effects of the stress, since comparable levels of myeloid progenitors were found in the bone marrow of both CVE/stress and control mice. Moreover, the sera from stressed mice pretreated with CVE further increased the CFU-GM formation. On the contrary, the spleen seemed to be less sensitive to acute stress in our experimental conditions. These findings are in line with our previous reports showing that the stress-induced reduction in bone marrow CFU-GM of rats exposed to electric shocks is mediated by activation of the HPA axis and by secretion of opioid agonists. No changes were observed in bone marrow, spleen and thymus total cell counts, and in relative organ weights. However, a 50% reduction in the body weight loss produced by the stress was observed in mice given the extract.

Titanocene modulation of cytokine imbalance induced by Ehrlich ascites tumour progression.

In the present work, we have studied the effects of two titanocenes, biscyclopentadienyldichlorotitanium IV (DDCT) and its derivative, biscyclopentadienylditiocianatetitanium IV (BCDT), on the production of cytokines [interferon-gamma (IFN-gamma), interelukin-1, interleukin (IL) 2, IL-4, and IL-10] by concanavalin A (Con A)-stimulated T cells obtained from Ehrlich ascites tumour (EAT)-bearing BALB/c mice. The treatment consisted of intraperitoneal (i.p) administration of 15 mg/kg/day DDCT for 2 days or 10 mg/kg/day BCDT for 3 days. We observed that the levels of IFN-gamma, but not IL-2, were dramatically increased in the early phase of EAT development. With tumour evolution, however, a sharp and progressive decrease in the levels of both IFN-gamma and IL-2 was found concomitantly to an enhancement in the levels of IL-10. Treatment of these mice with both titanocene compounds demonstrated that DDCT is more effective in modulating the cytokine imbalance induced by the tumour since it could prevent the early enhancement of IFN-gamma, the late decline of IFN-gamma and IL-2, and the increase in the IL-10. The administration of BCDT, in spite of preventing early IFN-gamma enhancement and increase in IL-10, did not produce any change in the IL-2 levels and did not prevent the decline of IFN-gamma levels during tumour evolution. Collectively, these results reveal that the ability of titanocenes to reverse tumour-induced immunosuppression and delay tumour growth is more evident in the DDCT compound, thus indicating that the substitution of the halides halogens by pseudohalogens, present in the molecular structure of BCDT, leads to a less effective antitumoral compound.

Ehrlich ascites tumor as a tool in the development of compounds with immunomodulatory properties.

In previous works, we have demonstrated that the myeloprotective properties of several natural and synthetic compounds are partly responsible for their antitumor activity in the Ehrlich ascites tumor (EAT) model. In this work, we present information that may be useful to the study of pharmacological and toxicological properties of compounds that affect the hematological compartment. Clonogenic studies in EAT-inoculated mice demonstrated a rapid decrease in bone marrow CFU-GM, whereas a progressive increase in splenic CFU-GM and cellularity was observed, followed by splenomegaly. Bone marrow cellularity declined on the third day after tumor challenge, returning to normal values thereafter. Serum from EAT-bearing mice produced detectable colony-stimulating activity in vitro. Similar results were observed with the conditioned medium from Ehrlich tumor cell cultures, but not with the cell-free Ehrlich tumor ascitic fluid. Tumor inoculation also resulted in a more striking depletion in the number of non-adherent cells in long-term bone marrow cell cultures (LTBMCs) with no bone marrow stroma formation. We speculate that the physiological alterations induced by the EAT growth can be used to assess the ability of compounds to modulate the hematopoietic response.

Enhancement of natural killer cell function by titanocenes in mice bearing Ehrlich ascites tumour.

In the present work, we studied the effects of two titanocenes, biscyclopentadienyldichlorotitanium IV, (DDCT) and its derivative, biscyclopentadienylditiocianatetitanium IV (BCDT), on the activity of natural killer (NK) cells in Ehrlich ascites tumour (EAT)-bearing BALB/c mice. In order to investigate a more direct effect of these compounds on NK cell function, we performed experiments with severe combined immunodeficiency (SCID) mice, which exhibit a normal NK cell response in the absence of T and B cells. The treatment consisted of intraperitoneal (i.p.) administration of 15 mg/kg/day of DDCT for 2 days or 10 mg/kg/day of BCDT for 3 days. In addition, to verify whether the effects produced by the titanocenes were compound specific or related to a direct antitumour effect, we also investigated the effects of a 3-day treatment with 100 mg/kg of cyclophosphamide cyclophosphamide on NK cell activity. Our results demonstrated that, in BALB/c and SCID mice, NK cell function declined to subnormal levels after inoculation of the tumour. In these animals, although treatment with DDCT and BCDT significantly enhanced NK cell function, only DDCT restored NK cell activity to normal values in all stages studied. Conversely, treatment with cyclophosphamide reduced NK cell function in nontumour bearing SCID mice and was also unable to restore the decreased NK activity of tumour-bearing SCID mice, thus demonstrating that the enhancement of NK cell function by titanocenes is compound specific. The same effect of cyclophosphamide was observed with BALB/c mice. In the present study, the up-modulatory effects of these two compounds on NK cell function reveal a new aspect of the mechanism of antitumoural action of titanocenes.